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The alveolar surface of the lung is lined by an epithelium consisting of type I (AECI) and type II alveolar epithelial cells (AECII). This epithelium is covered by a liquid alveolar lining layer (ALL). Besides intra‐alveolar surfactant, ALL also contains the alveolar epithelial glycocalyx on the apical side of AECI and AECII. To better understand the alveolar epithelial glycocalyx, its ultrastructural visualization by transmission electron microscopy is required. The aim of this study was to systematically re‐evaluate routine cytochemical methods for visualization of the alveolar epithelial glycocalyx and specifically its glycan components. For this purpose, we used chemical fixation by vascular perfusion with aldehydes as a common routine approach in mice. After fixation, staining is needed for glycocalyx visualization. Cytochemical staining agents such as alcian blue, ruthenium red, and lanthanum nitrate were compared. In addition, SNL (Sambucus nigra lectin) and UEA1 (Ulex europaeus agglutinin I) were used for sialic acid and fucose‐specific labeling. Alcian blue showed the strongest staining, with cloud‐like structures, whereas ruthenium red appeared as thread‐like structures. On the other hand, lanthanum nitrate did not stain the alveolar epithelial glycocalyx. For specific sialic acid and fucose labeling, both lectins presented a specific signal. In conclusion, these methods can be used routinely for assessing ultrastructural changes of the alveolar epithelial glycocalyx in experimental in vivo models under different physiological and pathological conditions. In addition, cytochemical staining by tissue massage and post‐embedding lectin labeling after vascular perfusion support 3R (reduction, refinement, replacement) principles of animal welfare. [ABSTRACT FROM AUTHOR]

Introduction: Various types of metaplastic changes, such as gastric metaplasia (foveolar type or antral type) and intestinal metaplasia, are observed in chronic cholecystitis but not in normal mucosa. Helicobacter species can be detected in the bile and gallbladder tissue of patients with benign gallbladder disease. Objective: The present study aimed to observe the correlation between chronic cholecystitis and the presence of Helicobacter pylori as well as the different types of metaplastic changes in gallbladder mucosa. Materials and Methods: This descriptive, observational study was conducted at the Departments of Pathology and Surgery at a tertiary care medical college and hospital from April 2021 to March 2022, using a cross-sectional design. All confirmed cases of chronic cholecystitis, with or without cholelithiasis, were included in the study. Results: Intestinal metaplasia was the most common type of metaplastic change in the gallbladder epithelium in chronic cholecystitis in this region. PAS and Alcian Blue positivity were significantly higher in cases with metaplasia compared to those without metaplasia. H. pylori was detected in 3 cases (1.55%) of chronic cholecystitis in this study population. Conclusion: Mucin histochemistry may help identify early metaplastic changes in gallbladder epithelium. The use of a combination of methods for the detection of H. pylori in gallbladder tissue may increase the detection of positive cases.

Background: The present study aimed at determining histological structure and histochemical properties of the henna partridge (Alectoris chukar) Harderian gland periodically. Objectives: For this purpose, the Harderian glands of 12 (6 females + 6 males) healthy henna partridges aged 3 and 6 months were used. Methods: Totally removed Harderian gland tissue samples were kept in 10% neutral formolin for 36 hours and fixed. After washing, dehydrating, and polishing with known histological techniques, tissue pieces were blocked in paraffin. Results: We found that a thin connective tissue capsule surrounds the Harderian gland of the henna partridge and that the capsule sends septums into the gland and divides the gland into lobes and lobules. It was observed that the corpus glandulae consist of low or high prismatic epithelial cells. Within each lobe, there is a single main draining channel which has a very large lumen along with primary draining channels. Primary and main draining channel epithelial cells have a single-layered cuboidal structure. By Gordon-Sweet staining, it was found that reticular fiber bundles started from the connective tissue capsule that surrounded the organ and spread by branching. The reticular fiber bundles extended in thin strands to the periphery of the corpus glandulae. These reticular fiber bundles in the regions where the connective tissue septums widened are thicker and form a network. The reticular fiber bundles are seen in the basal part of the epithelial cells forming the main draining channel and especially in the crypt areas. Reticular fibers are also found around blood vessels. Strong Alcian blue (AB)-positive epithelial cells are observed in the corpus glandulae in AB pH=2.5 staining, but no periodic acid-Schiff (PAS)- positive epithelial cells are observed in PAS staining. While the majority of primary draining channel cells show a strong AB-positive reaction, a few cells showed a weak PAS-positive reaction. Regarding the main draining channel epithelium, goblet cells show a weak and strong AB-positive reaction in AB pH=2.5 staining method, respectively and a weak PAS-positive reaction in PAS staining method. Conclusion: In line with these findings, no significant difference was found in terms of general histological structure and histochemical properties of the 3- and 6-month-old henna partridge Harderian gland. [ABSTRACT FROM AUTHOR]

Background and Objectives: Mucin has been implicated via various mechanisms in the development and growth of tumour cells. However, mucin expression studies in salivary gland tumours are limited, especially with samples from minor salivary glands. This study aims to investigate and compare mucin expression in benign and malignant salivary gland tumours of minor and major salivary gland origins. Materials and Methods: Special stains were used to stain neutral mucin (Periodic acid Schiff), sialomucin (Alcian Blue) and sulfomucin (Aldehyde Fuschin) within tissues from six normal salivary glands and 73 salivary gland tumours including 31 pleomorphic adenomas, 27 mucoepidermoid carcinomas, and 15 adenoid cystic carcinomas. A semi-quantitative approach was used to evaluate mucin expression within ductal lumens. Sialomucin was the most expressed mucin in all salivary gland tumours, regardless of origin. Results: A significant difference was observed in the mucin expression between benign and malignant salivary gland tumours, as pleomorphic adenoma showed three times significantly higher expression of sialomucin compared to mucoepidermoid carcinoma and adenoid cystic carcinoma (p = 0.028). Pleomorphic adenomas of major glands showed 42 times significantly higher expression of sialomucin compared to those of minor glands (p = 0.000). Conclusions: Sialomucin content in pleomorphic adenomas of major glands was vastly increased compared to that in minor glands. Differential sialomucin expression in benign and malignant salivary gland tumours suggests a role in diagnosing of borderline salivary gland tumours. [ABSTRACT FROM AUTHOR]

Background and Objective: In endometrial cytology, differentiating endometrial glandular stromal breakdown (EGBD) from endometrial endometrioid carcinoma (G1-EEC) is often difficult. In this study, we provided a new focus on chondroitin sulfate (CS), a major substrate component of the endometrial stroma, and assessed the diagnostic utility of Alcian Blue (AB) staining in the differential diagnosis in liquid-based cytological (LBC) samples. Materials and Methods: LBC specimens from 19 patients with a proliferative endometrium, 36 with EGBD, and 30 with G1-EEC who underwent endometrial cytology were stained with AB (pH 1.0), and their reactivity was observed. In addition, immunocytochemical staining of CS and CD31 was performed for five cases each to evaluate their interrelationship with blood vessels. Results: Regarding the 30 G1-EEC cases, at least one of the three representative staining patterns was observed by AB staining: dot-like, microtubular, and finely branched linear patterns. Moreover, the inner portion of the tubular material observed by AB staining expressed CD31. Conversely, in the 36 EGBD cases, only five metaplastic clusters with irregular protrusions and condensed stromal clusters (CSCs) showed a dot-like positive pattern, and background CSCs did not show reactivity to AB staining in any of the cases. Furthermore, the vascular structure expressing CD31 in cell clusters was also unclear. Conclusions: We demonstrated that AB staining shows different staining patterns in G1-EEC and EGBD, reflecting their different tissue structures. Our data provide new insights into endometrial cell diagnosis changes and demonstrate that AB staining is a potential new diagnostic aid tool for the differentiation of G1-EEC from EGBD.

Imaging mass cytometry (IMC) is a metal mass spectrometry-based method allowing highly multiplex immunophenotyping of cells within tissue samples. However, some limitations of IMC are its 1-µm resolution and its time and costs of analysis limiting respectively the detailed histopathological analysis of IMC-produced images and its application to small selected tissue regions of interest (ROI) of one to few square millimeters. Coupling on a single-tissue section, IMC and histopathological analyses could permit a better selection of the ROI for IMC analysis as well as co-analysis of immunophenotyping and histopathological data until the single-cell level. The development of this method is the aim of the present study in which we point to the feasibility of applying the IMC process to tissue sections previously Alcian blue-stained and digitalized before IMC tissue destructive analyses. This method could help to improve the process of IMC in terms of ROI selection, time of analysis, and the confrontation between histopathological and immunophenotypic data of cells. [ABSTRACT FROM AUTHOR]

Intestinal homeostasis involves the collaboration of gut barrier components, such as goblet cells and IgA-microbiota complexes, that are under the control of stress that promotes inflammatory responses addressed primarily in the colon. The aim of this study was to evaluate the effect of stress on mucins, goblet cells, and proinflammatory parameters in the proximal and distal regions of the small intestine. A group (n = 6) of female 8-week-old BALB/c mice underwent board immobilization stress (2 h per day for 4 days) and were sacrificed with isoflurane. Samples from proximal and distal small segments were collected to analyze the following: 1) goblet cells stained with periodic acid-Schiff (PAS) and with alcian blue (AB) to visualize histologically neutral and acidic mucins, respectively; 2) IgA-microbiota complexes identified by flow cytometry in intestinal lavages; and 3) MUC2, MUC5AC, and IL-18 mRNA levels in whole mucosal scrapings by reverse transcription-qPCR. Regarding the unstressed group, in the proximal region of small intestine both PAS+ and AB+ goblet cells were unchanged; however, MUC5AC and IL-18 mRNA levels were increased, and the percentage of IgA-microbiota complexes was reduced. In the distal segment, the number of PAS+ goblet cells was increased, whereas the number of AB+ goblet cells was reduced and did not affect the remaining parameters. The data suggest that stress induces inflammation in the proximal small intestine; these findings may provide an experimental reference for human diseases that may affect the proximal small intestine, such as Crohn's disease, in which stress contributes to the progression of intestinal inflammation or relapse. [ABSTRACT FROM AUTHOR]

Background : Acupuncture, practiced for millennia, lacks a clear anatomical definition for acupoints. A prevailing theory suggests that acupoints overlap with skin areas with higher mast cell density. Skin spots stained with intravenously infused Evans blue (EB), indicative of neurogenic inflammation, have recently been posited as acupoints in rats. Objectives: To demonstrate the concordance between EB-reactive skin spots and mast cell-enriched acupoints. Methods : We employed staining and RNA-seq analysis to delineate the morphological characteristics and gene expression profiles of EB-reactive skin spots in rats. Results : EB infusion revealed a novel nodal structure on the rat skin surface, visible to the naked eye, with dimensions of approximately 1 mm in both diameter and height. Around 30 such nodes were identified on one side of the abdominal area, spaced roughly 3 mm apart, excluding the linea alba. RNA-seq analysis indicated that the gene expression patterns within these nodes markedly differed from both non-nodal skin areas and lymph nodes. Histological examination using toluidine blue revealed a significantly greater mast cell count in the nodes than in non-nodal skin regions. Additionally, the nodes stained positively with Alcian blue and Hemacolor, reagents known to mark primo vascular tissues. Conclusion : Our findings suggest that EB-reactive nodes are indeed rich in mast cells. Further research is warranted to establish these skin nodes as surface primo nodes.

The kinetochore links chromosomes to spindle microtubules to drive chromosome segregation at cell division. While we know nearly all mammalian kinetochore proteins, how these give rise to the strong yet dynamic microtubule attachments required for function remains poorly understood. Here, we focus on the Astrin-SKAP complex, which localizes to bioriented kinetochores and is essential for chromosome segregation but whose mechanical role is unclear. Live imaging reveals that SKAP depletion dampens the movement and decreases the coordination of metaphase sister kinetochores and increases the tension between them. Using laser ablation to isolate kinetochores bound to polymerizing versus depolymerizing microtubules, we show that without SKAP, kinetochores move slower on both polymerizing and depolymerizing microtubules and that more force is needed to rescue microtubules to polymerize. Thus, in contrast to the previously described kinetochore proteins that increase the grip on microtubules under force, Astrin-SKAP reduces the grip, increasing attachment dynamics and force responsiveness and reducing friction. Together, our findings suggest a model where the Astrin-SKAP complex effectively "lubricates" correct, bioriented attachments to help preserve them.

Background: Obesity affects multiple organs in the body including salivary glands resulting in secretory dysfunction. The selective water channels, aquaporins (AQPs) have a role in salivary secretion. As far as the authors know, no previous research has been done to investigate the effect of a high fat diet on AQ8 localization in sublingual glands (SLGs). Methods: 12 male Sprague-Dawley albino rats were equally divided into two groups: a control group and a high fat diet (HFD) group. After 12 weeks, their blood glucose, insulin, serum triglyceride, cholesterol, HDL and LDL were evaluated. SLGs were dissected out and stained by H&E, PAS/Alcian and immunohistochemically using anti-smooth muscle actin (a SMA) and anti-AQ8 antibodies. Results: Compared to the control group, blood insulin resistance, serum triglyceride, cholesterol, LDL levels were highly significantly increased, while HDL was highly significantly decreased. HFD SLGs were distorted with vacuolations, thick fibrous septa and congested blood vessels. Diameters were decreased. Image analysis of PAS/Alcian and a SMA showed a highly significant increase in the (alcian blue) and a significant increase in the area % of a SMA, while the expression of AQ8 was highly significant decreased compared to the control group. Conclusions: The decreased AQ8 immunoexpression in HFD rats might contribute to gland 'secretory dysfunction. [ABSTRACT FROM AUTHOR]

Imaging mass cytometry (IMC) is a metal mass spectrometry-based method allowing highly multiplex immunophenotyping of cells within tissue samples. However, some limitations of IMC are its 1-µm resolution and its time and costs of analysis limiting respectively the detailed histopathological analysis of IMC-produced images and its application to small selected tissue regions of interest (ROI) of one to few square millimeters. Coupling on a single-tissue section, IMC and histopathological analyses could permit a better selection of the ROI for IMC analysis as well as co-analysis of immunophenotyping and histopathological data until the single-cell level. The development of this method is the aim of the present study in which we point to the feasibility of applying the IMC process to tissue sections previously Alcian blue-stained and digitalized before IMC tissue destructive analyses. This method could help to improve the process of IMC in terms of ROI selection, time of analysis, and the confrontation between histopathological and immunophenotypic data of cells.

Background and Objectives: Mucin has been implicated via various mechanisms in the development and growth of tumour cells. However, mucin expression studies in salivary gland tumours are limited, especially with samples from minor salivary glands. This study aims to investigate and compare mucin expression in benign and malignant salivary gland tumours of minor and major salivary gland origins. Materials and Methods: Special stains were used to stain neutral mucin (Periodic acid Schiff), sialomucin (Alcian Blue) and sulfomucin (Aldehyde Fuschin) within tissues from six normal salivary glands and 73 salivary gland tumours including 31 pleomorphic adenomas, 27 mucoepidermoid carcinomas, and 15 adenoid cystic carcinomas. A semi-quantitative approach was used to evaluate mucin expression within ductal lumens. Sialomucin was the most expressed mucin in all salivary gland tumours, regardless of origin. Results: A significant difference was observed in the mucin expression between benign and malignant salivary gland tumours, as pleomorphic adenoma showed three times significantly higher expression of sialomucin compared to mucoepidermoid carcinoma and adenoid cystic carcinoma (p = 0.028). Pleomorphic adenomas of major glands showed 42 times significantly higher expression of sialomucin compared to those of minor glands (p = 0.000). Conclusions: Sialomucin content in pleomorphic adenomas of major glands was vastly increased compared to that in minor glands. Differential sialomucin expression in benign and malignant salivary gland tumours suggests a role in diagnosing of borderline salivary gland tumours.

Carcinoid tumours of the appendix are low-grade neoplasms with neuroendocrine differentiation. They are typically found incidentally during appendectomies. Tubular carcinoid is an extremely rare, distinct, and benign form of appendiceal carcinoid. Here, the authors present the case of a 32-year-old male who presented with an abdominal mass and pain radiating to the right iliac fossa. Appendectomy was performed, and the gross examination revealed a congested and exudate-covered appendix. Microscopic analysis showed small tubules infiltrating the appendix wall, along with evidence of acute appendicitis. Differential diagnoses considered included carcinoid tumour with glandular differentiation and metastatic adenocarcinoma. Additional sections were examined, and a final diagnosis of tubular carcinoid was made after conducting a panel of immunohistochemical markers and Alcian blue stain. Since most tubular carcinoids are not identifiable on gross, microscopic assessment and serial sectioning play a crucial role in diagnosing this condition.

Summary: We present here a modified protocol for safranin-alcian blue staining of paraffin-embedded plant samples. After deparaffination, the double staining uses a 2:1 mix of 1% safranin and 1% alcian blue. This method is less expensive than previously published double staining protocols as it does not require absolute ethanol. Moreover, safranin-alcian blue staining creates a range of hues that allows good cell type differentiation, it gives reproducible results for a wide variety of plant tissues and is easy to use. We hope it will be found useful both for research and teaching purposes. [ABSTRACT FROM AUTHOR]

Introduction. Losing of small tissues during tissue preparatory steps may seriously affect pathological diagnostic performance. Using an appropriate tissue marking dye could be an alternative solution. Therefore, the aim of the study was to find a suitable tissue marking dye to enhance the observable ability of various types of small-size tissues during several steps of tissue preparation. Material and methods. Various small-size samples of various organs and tissues (0.2 to 0.3 cm), including breast, endometrial, and cervical tissue, stomach, small and large intestine, lung, and kidney, were marked with different dyes such as merbromin, hematoxylin, eosin, crystal violet, and alcian blue prior to tissue processing step and their colored-observable ability was evaluated by pathology assistants. Moreover, the diagnostic interfering effect of each tissue marking dye was determined by pathologists. Results. Merbromin, hematoxylin, and alcian blue increased the colored-observable ability of small tissue samples. We suggest using hematoxylin as a tissue marking dye over merbromin and alcian blue because of less toxicity and no interference effect in the step of routine pathological slide examination. Conclusions. Hematoxylin could be a suitable tissue marking dye for small-size samples and may improve the preanalytical process of tissue preparation in pathological laboratories. [ABSTRACT FROM AUTHOR]

Aim. To compare efficiency and specific features of transthyretin amyloid staining by different histological dyes and thus to assess their suitability for diagnostic purposes. Materials and methods. Samples of left and right heart ventricles were taken from patients over 70 years-old of both genders (n=10) with immunohistochemically verified transthyretin amyloidosis (ATTR). All samples were stained with Congo red, Alcian blue, toluidine blue and methylene violet. Results. Specificity and sensitivity of Congo red staining was comparable to those of immunohistochemical staining. For verification of amyloid presence after Congo red staining one could use fluorescent microscopy instead of polarization microscopy. It allows a more accurate diagnosis of amyloidosis. Confocal microscopy with spectral unmixing improves detection sensitivity of amyloid by elimination of background fluorescence of muscle tissue and autofluorescence of lipofuscin. Alcian blue staining gives the same result as Congo red. In addition, its less labor-intensive and free of false-positive and false-negative results caused by final processing of slide preparation. Toluidine blue and methylene violet develop metachromatic staining upon binding to transthyretin fibrils, likely due to specific biochemical features of these fibrils. Conclusion. The most reliable method for histochemical diagnosis of ATTR is the Congo red staining with subsequent analysis using fluorescence or confocal microscopy. For diagnostic screening, the use of Sodium sulphate-Alcian blue staining method is highly promising. Metachromatic stains are less effective for ATTR diagnosis.

Superficial angiomyxoma (SA) is a benign tumor characterized by extensive myxoid stroma, numerous small blood vessels, sparse spindle-shaped fibroblastic cells, and inflammatory cell infiltrate. Oral cavity SA is extremely rare and typically presents as a painless, slow growth. We experienced SA in the mandibular gingiva that is rapidly growing. The patient was a 15-year-old female whose chief complaint was a painless mass in the lingual gingiva of the mandible that increased in size over 1 month. An excisional biopsy was performed under local anesthesia. According to histopathological examination, the mass was diagnosed as SA. The patient experienced recurrence twice because of positive margins. The second recurrent lesion, including periosteum, was resected, and no recurrence has been observed for 1 year. The cause of rapid growth was attributed to edematous changes due to tongue habit or traumatic stimuli. As this case exhibited repeated local recurrence, careful follow-up is required. [ABSTRACT FROM AUTHOR]

Aims: Minimally manipulated cells, such as autologous bone marrow concentrates (BMC), have been investigated in orthopaedics as both a primary therapeutic and augmentation to existing restoration procedures. However, the efficacy of BMC in combination with tissue engineering is still unclear. In this study, we aimed to determine whether the addition of BMC to an osteochondral scaffold is safe and can improve the repair of large osteochondral defects when compared to the scaffold alone. Methods: The ovine femoral condyle model was used. Bone marrow was aspirated, concentrated, and used intraoperatively with a collagen/hydroxyapatite scaffold to fill the osteochondral defects (n = 6). Tissue regeneration was then assessed versus the scaffold-only group (n = 6). Histological staining of cartilage with alcian blue and safranin-O, changes in chondrogenic gene expression, microCT, peripheral quantitative CT (pQCT), and force-plate gait analyses were performed. Lymph nodes and blood were analyzed for safety. Results: The results six months postoperatively showed that there were no significant differences in bone regrowth and mineral density between BMC-treated animals and controls. A significant upregulation of messenger RNA (mRNA) for types I and II collagens in the BMC group was observed, but there were no differences in the formation of hyaline-like cartilage between the groups. A trend towards reduced sulphated glycosaminoglycans (sGAG) breakdown was detected in the BMC group but this was not statistically significant. Functional weightbearing was not affected by the inclusion of BMC. Conclusion: Our results indicated that the addition of BMC to scaffold is safe and has some potentially beneficial effects on osteochondral-tissue regeneration, but not on the functional endpoint of orthopaedic interest.

In the wake of the COVID-19 pandemic, fungal infections of the maxillofacial region have become prevalent, making their accurate diagnosis vital. Histopathological staining remains a simple, cost-effective technique for differentiation and diagnosis of the causative fungal organisms. The present study aims to evaluate the staining efficacy of Periodic Acid-Schiff (PAS), Alcian Blue, Safranin-O and Gomori's Methenamine Silver (GMS) on fungal smears. This research work also attempts to study the morphometric characteristics of Candida albicans, Aspergillus flavus, Rhizopus oryzae. Candida albicans, Aspergillus flavus and Rhizopus oryzae, 10 smears each, were stained using PAS, Alcian Blue, Safranin-O and GMS. The morphological characteristics and staining efficacy were examined, and semi-quantitative scoring was performed. Candida albicans, Aspergillus flavus and Rhizopus oryzae were stained for the first time with Safranin-O. The morphometric traits were then analyzed using an image analysis software. Safranin-O provided the most reliable staining efficacy amongst the stains and optimum morphological definition for all three organisms. Safranin-O was found to be superior to PAS and GMS, ensuring detection of even the most minute mycotic colonies. The hyphae of Aspergillus flavus to be the largest, and the spores and fruiting body of Rhizopus oryzae were found to be the largest amongst the three organisms compared. Early and accurate diagnosis of fungal infections can significantly reduce morbidity in orofacial fungal infections. [ABSTRACT FROM AUTHOR]

Transparent exopolymer particles (TEPs) and their precursors play a critical role in various environmental processes, including contaminant transport and membrane fouling. This study presents an improved Alcian blue (AB) staining-based TEP measurement method and investigates spectroscopic proxies to enhance analysis convenience and reproducibility. The proposed method employs deionized (DI) water and sonication to extract particulate TEPs (TEP 0.4μm) and particulate TEPs and their precursors (TEP 10kDa), thus eliminating the need for concentrated sulfuric acid and simplifying the procedure with an integrated standard curve. This AB-DI method had a low detection limit of 5 μg X eq /L for a 1 L sample volume, allowing precise measurements to be made across a broad concentration range. When used in wastewater treatment and microfiltration membrane filtrates, the AB-DI method provided insights into the removal mechanisms of TEPs and their precursors. This study also identified effective spectroscopic proxies by correlating TEP concentrations with optical properties, achieving remarkable estimation accuracy using the UV 254 absorbance of TEP-extracted solutions. The UV 254 and fluorescence intensities of the protein-like peaks in raw water samples demonstrated significant potential as proxies for TEPs and their precursors. The proposed AB-DI method represents a viable approach for the real-time TEP monitoring for desalination and water reuse processes. [Display omitted] [ABSTRACT FROM AUTHOR]

Background: Histological staining methods for Cryptococcus identification vary in accuracy. This study aimed to investigate the clinical value of Grocott methenamine silver (GMS), periodic acid-Schiff (PAS), and Alcian blue (AB) staining in the diagnosis of pulmonary cryptococcosis (PC). Methods: From April 2004 to June 2021, the clinical and pathological data of 152 patients with PC were collected from the Department of Pathology, Sun Yat-sen University Cancer Center. The sensitivity and identifiability of GMS, PAS, and AB staining for histological diagnosis were systematically evaluated using statistical methods combined with the microscopic characteristics of PC cases. Results: Statistical analysis showed that the detection rates of GMS, PAS, and AB staining were 100.0% (152/152), 94.7% (144/152), and 81.6% (124/152), respectively. McNemar's test showed that the sensitivity of GMS was significantly higher than those of PAS (P = 0.008) and AB stains (P < 0.001). Both PAS and AB stains had obvious non-specific staining, which interfered with the detection of Cryptococcus , and increased diagnostic difficulties. In contrast, in GMS staining, Cryptococcus spores were prominent with a clean background and were clearly observed at low or medium power magnification, with the identifiability significantly better than those of PAS or AB staining. Conclusion: GMS staining had sensitivity up to 100%, and identifiability that was better than those of PAS and AB staining. GMS is the best method for histological diagnosis of PC. [ABSTRACT FROM AUTHOR]

Automated methods exist for double staining bone and cartilage in fetal rat, rabbit and mice using alizarin red S and Alcian blue. None of the published methods produce consistently stained bone and cartilage in mouse fetuses. Consequently, we modified an automated staining method for use with mouse fetuses. Methanol replaced 95% industrial denatured alcohol, and 5% glacial acetic acid was used instead of 20%. We describe here our new protocol, which has been used successfully to stain specimens for regulatory prenatal developmental toxicity studies. [ABSTRACT FROM AUTHOR]

Introduction: Histological diagnosis of Barrett's oesophagus (BE) in mucosal biopsies is challenging and affected by multiple factors. Goblet Cells (GCs) are not distributed uniformly in BE and is dependent on sampling probabilities. Furthermore, GC Mimickers (GCM) are potential pitfalls in the diagnosis of Intestinal Metaplasia (IM). Alcian Blue (AB) stain has been extensively used in detection of GC's although it has the limitation of low specificity with positive staining for GCM. Recently, CDX2 Immunohistochemistry (IHC) is reported to be highly sensitive and specific marker which has shown to identify early intestinal phenotype even in absence of diagnostic GCs and especially pertaining to conditions where characteristic morphological changes are not apparent. Aim: To study the histomorphology of non neoplastic and neoplastic lesions of the Gastroesophageal Junction (GEJ) in reflux patients and evaluate the diagnostic role of CDX2 IHC versus AB stain in detecting IM. Materials and Methods: This retrospective study was conducted in Department of Pathology at Kasturba Medical College, Manipal, Karnataka, India, on 55 patients with clinical features of reflux and adequate records of GEJ biopsies, diagnosed over 6 years from January 2012 to August 2018. Clinical presentation, endoscopic findings and histomorphology (18 parameters) were recorded. AB stain and CDX2 (IHC) were performed and evaluated in all cases. A detailed histological evaluation was done for all cases and subsequently, sensitivity, specificity, positive predictive value and negative predictive value of CDX2 IHC to identify early intestinal differentiation was calculated. Results: Of 55 cases, 28 were BE, 19-Reflux oesophagitis (RE) and 8-adenocarcinoma. Heart burn and chest pain were the most common clinical presentations of BE. Endoscopy of BE predominantly showed hiatus hernia with tongue like projections of the gastric mucosa. Histologically, intraepithelial eosinophils and spongiosis were more common features in RE. Barrett's oesophagus showed columnar epithelium with multilayering, presence of IM with GC (1-20/crypt) along with sub-squamous buried epithelium and splitting of muscularis mucosa. By IHC, as compared to AB; CDX2 IHC was more sensitive (100% vs 78.2%) and specific (96.5% vs 82.6%) for detecting an intestinal phenotype. The five cases (22%) of BE contained only GCM in the biopsy, were CDX2 negative but showed a false positivity for AB. In BE, CDX2 additionally highlighted positivity in non GC columnar cells which were AB negative. The CDX2 showed diffuse positivity in dysplasia with focal strong to absent expression in adenocarcinoma. Conclusion: The CDX2 efficiently differentiated between GC and pseudo GC. Its presence in the absence of AB in non GC columnar cells suggests that it effectively detects intestinal phenotypic features even before morphological features are evident. [ABSTRACT FROM AUTHOR]

In the realm of inflammation, including conditions like cancers, infections, and autoimmune diseases, the emergence of tertiary lymphoid structures (TLS) holds significant implications for prognosis and treatment response. Yet, traditional methodologies such as immunohistochemistry and immunofluorescence falter in capturing the nuanced complexities of TLS, necessitating innovative approaches, like imaging mass cytometry (IMC), to unravel their significance. With the capacity to concurrently assess nearly 40 markers within the same tissue section, IMC transcends the constraints of traditional approaches.This chapter delineates the IMC methodology tailored for TLS visualization and subsequent quantification. By leveraging IMC on formalin-fixed, paraffin-embedded (FFPE) sections of human colorectal cancer (CRC) tissues, we aim to establish a standardized protocol that elucidates the intricacies of TLS dynamics and composition., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

ABSTRACT Introduction The classification of intestinal metaplasia by histochemistry methods has been described as the most appropriate. However, current algorithms are not replicable in laboratories due to severe limitations. Objective To evaluate a new algorithm to differentiate histochemical types of intestinal metaplasia. Material and Method: Cross-sectional research in which 512 gastric biopsies with intestinal metaplasia (paraffin blocks) were evaluated by a new algorithm using two types of Alcian Blue dyes during February-March of 2020 in the pathological anatomy service of the Maria Auxiliadora Hospital, Lima, Peru. This evaluation consisted of two steps: visualization of acid mucins in the columnar cells of the gastric mucosa and calculation of the weighted Kappa statistic. Results Histochemical types of intestinal metaplasia showed as follows: Type I, 398 (77.7%); Type II, 81 (15.8%) and Type III, 33 (6.5%). The weighted Kappa statistic was 0.79 (p

BackgroundHistological staining methods for Cryptococcus identification vary in accuracy. This study aimed to investigate the clinical value of Grocott methenamine silver (GMS), periodic acid-Schiff (PAS), and Alcian blue (AB) staining in the diagnosis of pulmonary cryptococcosis (PC).MethodsFrom April 2004 to June 2021, the clinical and pathological data of 152 patients with PC were collected from the Department of Pathology, Sun Yat-sen University Cancer Center. The sensitivity and identifiability of GMS, PAS, and AB staining for histological diagnosis were systematically evaluated using statistical methods combined with the microscopic characteristics of PC cases.ResultsStatistical analysis showed that the detection rates of GMS, PAS, and AB staining were 100.0% (152/152), 94.7% (144/152), and 81.6% (124/152), respectively. McNemar’s test showed that the sensitivity of GMS was significantly higher than those of PAS (P = 0.008) and AB stains (P < 0.001). Both PAS and AB stains had obvious non-specific staining, which interfered with the detection of Cryptococcus, and increased diagnostic difficulties. In contrast, in GMS staining, Cryptococcus spores were prominent with a clean background and were clearly observed at low or medium power magnification, with the identifiability significantly better than those of PAS or AB staining.ConclusionGMS staining had sensitivity up to 100%, and identifiability that was better than those of PAS and AB staining. GMS is the best method for histological diagnosis of PC.

Introduction: Histological diagnosis of Barrett’s oesophagus (BE) in mucosal biopsies is challenging and affected by multiple factors. Goblet Cells (GCs) are not distributed uniformly in BE and is dependent on sampling probabilities. Furthermore, GC Mimickers (GCM) are potential pitfalls in the diagnosis of Intestinal Metaplasia (IM). Alcian Blue (AB) stain has been extensively used in detection of GC’s although it has the limitation of low specificity with positive staining for GCM. Recently, CDX2 Immunohistochemistry (IHC) is reported to be highly sensitive and specific marker which has shown to identify early intestinal phenotype even in absence of diagnostic GCs and especially pertaining to conditions where characteristic morphological changes are not apparent. Aim: To study the histomorphology of non neoplastic and neoplastic lesions of the Gastroesophageal Junction (GEJ) in reflux patients and evaluate the diagnostic role of CDX2 IHC versus AB stain in detecting IM. Materials and Methods: This retrospective study was conducted in Department of Pathology at Kasturba Medical College, Manipal, Karnataka, India, on 55 patients with clinical features of reflux and adequate records of GEJ biopsies, diagnosed over 6 years from January 2012 to August 2018. Clinical presentation, endoscopic findings and histomorphology (18 parameters) were recorded. AB stain and CDX2 (IHC) were performed and evaluated in all cases. A detailed histological evaluation was done for all cases and subsequently, sensitivity, specificity, positive predictive value and negative predictive value of CDX2 IHC to identify early intestinal differentiation was calculated. Results: Of 55 cases, 28 were BE, 19-Reflux oesophagitis (RE) and 8-adenocarcinoma. Heart burn and chest pain were the most common clinical presentations of BE. Endoscopy of BE predominantly showed hiatus hernia with tongue like projections of the gastric mucosa. Histologically, intraepithelial eosinophils and spongiosis were more common features in RE. Barrett’s oesophagus showed columnar epithelium with multilayering, presence of IM with GC (1-20/crypt) along with sub-squamous buried epithelium and splitting of muscularis mucosa. By IHC, as compared to AB; CDX2 IHC was more sensitive (100% vs 78.2%) and specific (96.5% vs 82.6%) for detecting an intestinal phenotype. The five cases (22%) of BE contained only GCM in the biopsy, were CDX2 negative but showed a false positivity for AB. In BE, CDX2 additionally highlighted positivity in non GC columnar cells which were AB negative. The CDX2 showed diffuse positivity in dysplasia with focal strong to absent expression in adenocarcinoma. Conclusion: The CDX2 efficiently differentiated between GC and pseudo GC. Its presence in the absence of AB in non GC columnar cells suggests that it effectively detects intestinal phenotypic features even before morphological features are evident.

Mid1 and Mid2 are ubiquitin ligases that regulate microtubule dynamics and whose mutation is associated with X-linked developmental disorders. We show that astrin, a microtubule-organizing protein, co-purifies with Mid1 and Mid2, has an overlapping localization with Mid1 and Mid2 at intercellular bridge microtubules, is ubiquitinated by Mid2 on lysine 409, and is degraded during cytokinesis. Mid2 depletion led to astrin stabilization during cytokinesis, cytokinetic defects, multinucleated cells, and cell death. Similarly, expression of a K409A mutant astrin in astrin-depleted cells led to the accumulation of K409A on intercellular bridge microtubules and an increase in cytokinetic defects, multinucleated cells, and cell death. These results indicate that Mid2 regulates cell division through the ubiquitination of astrin on K409, which is critical for its degradation and proper cytokinesis. These results could help explain how mutation of MID2 leads to misregulation of microtubule organization and the downstream disease pathology associated with X-linked intellectual disabilities.

We investigated physicochemical characteristics of dye lots sold as "alcian blue" using the Biological Stain Commission (BSC) precipitation test, differential scanning calorimetry, high performance liquid chromatography, thin layer chromatography and UV/visible spectroscopy. Four blue phthalocyanine dyes were detected in 11 commercial dye lots. These four included the original ingrain blue 1 CI 74000 dye and the dye sold with the name "alcian blue pyridine variant"; we discuss also the possible identity of the additional two dyes. A proposed extension to the BSC analytic scheme is presented that could distinguish three categories of commercial alcian blue dyes from each other and from the original alcian blue 8G. [ABSTRACT FROM AUTHOR]

Sulfomucins are in some body locations and species a normal occurrence, whereas in other situations, are a sign of pathology. Sulfomucin content on histological sections and isolated material is frequently analyzed with Alcian blue staining at pH 1.0. However, since the stain detects the charge, a high density of other charged molecules, such as sialic acids, has potential to impede specificity. Here, we compared the outcome from four staining protocols with the level of sulfation determined by liquid chromatography–tandem mass spectrometric analysis on samples from various tissues with variable sulfation and sialylation levels. We found that a protocol we designed, including rinsing with MetOH and 0.5 M NaCl buffer at pH 1.0, eliminates the false positive staining of tissues outperforming commonly recommended solutions. In tissues with low-to-moderately sulfated mucins (e.g. human stomach and salmonid epithelia), this method enables accurate relative quantification (e.g. sulfate scoring comparisons between healthy and diseased tissues), whereas the range of the method is not suitable for comparisons between tissues with high sulfomucin content (e.g. pig stomach and colon). [ABSTRACT FROM AUTHOR]

Background and Aim: Direct observation, scanning electron microscopy (SEM) is a common method used for the observations of biofilms. N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide) (EDC) fixation method has proven to be a valuable fixation method in the observation of these biofilms. Still, it entails a method of biofilm fixation that can damage slim structures, leading to the impossible observation of biofilm development. In contrast, alcian blue and lysine (ABL) fixation technique appears more glycocalyx of biofilm, fully preserved samples, which may provide much insight into the development of B. subtilis biofilms. Materials and Methods: Here, the evaluation of the fixation of ABL technique for the study of B. subtilis biofilms was carried out in situ, on Congo red agar. In doing so, the comparison to commonly use conventional EDC technique for sample fixation, and observation was carried out. Observations were based on SEM over 30 samples. Results: Overall, ABL technique provided excellent observation of biofilms formed in situ, on Congo red agar, and revealed slime structures, which have not been observed, much in standard EDC fixation or earlier in other studies of these biofilms in B. subtilis. Conclusion: This study reported the appropriate use of ABL in the fixation technique for the preservation of biofilm of B. subtilis.

Oral focal mucinosis (OFM) is a unique benign lesion of the oral cavity with uncertain etiology which is analogous to cutaneous focal mucinosis. It mainly affects women in their fourth and fifth decades of life. The diagnosis of this condition is based on histopathological examination, as it lacks characteristic clinical and radiographic features. Its pathophysiology is associated with fibroblasts producing excessive amounts of hyaluronic acid, which causes localized myxomatous changes. Here, we describe the occurrence of this rare entity in a 54-year-old female patient involving attached gingiva of the left posterior mandibular region along with emphasis on its histopathological and histochemical findings to differentiate it from clinically and microscopically look-alike lesions., Competing Interests: Human subjects: Consent was obtained or waived by all participants in this study. Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work., (Copyright © 2024, Das et al.)

In vitro studies with cultured cells are often conducted as an important part of basic research. Adherent cells are typically cultivated in flasks or trays, for which cell staining and subsequent visualization become impractical. We here present a simple step-by-step method for growing adherent cells directly on glass microscope slides, using low-cost equipment readily available in most laboratories. Most parameters such as type of microscope slide (e.g. surface coating), cell seeding concentrations and incubation times can be adjusted according to cell line characteristics and experimental aims, reflecting the methods' flexibility. Through our experiments, microscope slides proved to provide an acceptable surface for cell adhesion and growth of the tested cell lines, as well as being robust and functional with respect to downstream procedures. The method can potentially be combined with different techniques for visualization of experimental effects, such as histological staining methods, fluorescent staining, and immunochemistry. In our method development we have successfully cultivated three different cell lines directly on microscope slides - Atlantic salmon kidney cells (ASK), rainbow trout gill cells (RTgill-W1), and human cancerous lung cells (A549) - and subjected them to various experimental treatments. Finally, as proof-of-concept we provide examples of successful histological staining of the fixed cells. Experimental design in short:•Cultivate cells and calculate cell concentration•Seed a small volume of growth medium with an appropriate number of cells on microscope slide in an area confined by hydrophobic marker•Let cells adhere over night before adding more growth medium or directly conducting experiments and fixing cells for downstream applications., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Author(s).)

Hyalinization is a process of conversion of stromal connective tissue into a homogeneous, acellular translucent material. Nevertheless, hyalinization could provide insights into the biologic behaviour and prognosis of pathological lesions. Few studies with limited sample size have intended to assess the correlation of hyalinization and biologic behaviour in oral lesions. The current review aims to comprehensively appraise the mechanism of hyalinization in pathological oral hyalinizing lesions (OHL) and its clinical implications with emphasis on differential stains employed. An electronic search was performed in the PubMed database (from year 2000–2020) using the keywords "special stains in oral hyalinizing lesions", "significance of hyalinization in oral lesions" and "hyalinization and biologic behaviour". Original research articles analyzing the effect of hyalinization on biologic characteristics of the lesion were evaluated in this review. Narrative review articles that provided insights into the mechanism of hyalinization and maturity of collagen fibers were also considered for analysis. The presence of hyalinization does seem to have a significant effect on the biologic behaviour of pathological lesions. There is substantial scope to further investigate the process of hyalinization on larger samples and its correlation with the aggressive behaviour of OHLs. Special stains and advanced investigations such as immunohistochemistry for stromal markers would define the nature of hyalinized material and validate the correlation. The prediction of the biologic behaviour of a lesion established through assessment of hyalinization would prevent unwanted over or under treatment leading to a better prognosis. [ABSTRACT FROM AUTHOR]

Glycocalyx (GCX) is a thin layer of negatively charged glycoproteins that covers the vascular endothelial surface and regulates various biological processes. Because of the delicate and fragile properties of this structure, it is difficult to detect GCX morphologically. We established a simple method for a three-dimensional visualization of endothelial GCX using low-vacuum scanning electron microscopy (LVSEM) on formalin-fixed paraffin-embedded (FFPE) sections. Mouse kidney tissue was fixed with 10% buffered formalin containing 1% Alcian blue (ALB) via perfusion and immersion. FFPE sections were observed by light microscopy (LM) and LVSEM, and formalin-fixed epoxy resin-embedded ultrathin sections were observed by transmission electron microscopy (TEM). The endothelial GCX from various levels of kidney blood vessels was stained blue in LM and confirmed as a thin osmiophilic layer in TEM. In LVSEM, the sections stained by periodic acid methenamine silver (PAM) revealed the endothelial GCX as a layer of dense silver-enhanced particles, in both the samples fixed via perfusion and immersion. Correlative light and electron microscopy (CLEM) revealed the fine visible structure of endothelial GCX. This simple method using FFPE samples with ALB will enable the three-dimensional evaluation of endothelial GCX alterations in various human diseases associated with endothelial injury in future studies. [ABSTRACT FROM AUTHOR]

Myxoid meningioma is a rare histological variant of meningiomas grouped into the subtype of metaplastic meningiomas (World Health Organization grade I) characterized by the presence of myxoid areas and meningothelial cells along with unique ultrastructural features that help distinguish it from other meningiomas. Hereby, we report a case of a myxoid meningioma in a 40-year-old female who presented with altered sensorium and loss of consciousness. Imaging studies showed a dura-based solitary mass located in the left frontal lobe. The tumor was excised completely. Histopathology revealed a benign appearing myxoid neoplasm with uniform elongated cells, without any atypia and a very low mitotic activity. Immunohistochemical stains showed positivity for vimentin and epithelial membrane antigen confirming the diagnosis of myxoid meningioma. Only seven cases of myxoid meningioma have been reported till date in the literature and ours is the eighth case.

The first step in almost every investigation of skeletal phenotypes is analysis of whole-mount skeletal preparations. Whole-mount skeletal staining permits evaluation of the shapes and sizes of skeletal elements in their appropriate locations. The technique is thus the major method for detecting changes in skeletal patterning. Because cartilage and bone can be distinguished by differential staining, this technique is also a powerful means to assess the pace of skeletal maturation. This protocol covers staining of the pre- and postnatal mouse skeleton using Alcian blue and Alizarin red to identify cartilage and bone, respectively.

Teaching skeletal system would be more attractive using real/preserved specimen compared to those using only book. The aim of the research was to develop clearing and staining method of animal skeleton specimen using Alizarin Red S-Alcian Blue as teaching aid tool. The specimen were eviscerated, fixed, stained, cleared, and keep in glycerine solution. The spesimen will increase effectiveness in elementary school science teaching and learning.

A 15-year-old captive female Urutu snake (Bothrops alternatus) was presented with anorexia, apathy, dehydration, pale mucous membranes and marked enlargement of the caudal third of the coelomic cavity. Radiographs and ultrasonography were suggestive of faecal impaction or neoplasia. Post-mortem findings consisted of solid faecal impaction of the intestines, secondary to obstruction caused by a rectal tumour. Cytological examination of impression smears of the tumour revealed polygonal to round neoplastic cells arranged in clusters in an abundant extracellular mucinous matrix. Histologically, there was transmural growth of neoplastic epithelial cells arranged in small nests or individually and immersed in abundant extracellular mucin lakes. The neoplastic cells were immunopositive for cytokeratin. Clinical and pathological aspects indicated a diagnosis of mucinous rectal adenocarcinoma. [ABSTRACT FROM AUTHOR]

The present study investigated the effect of replacement of fish meal and soybean meal with shrimp wastes meal on the intestine tissue in common carp Cyprinus carpio fingerlings. Five isonitrogenous (31.66±0.40) and isocaloric (405.57±5.35 kg / 100 g) diets were formulated including control diet (C), 25% and 50% of the total dietary protein replacement of fish meal (T1 and T2 respectively) or soybean meal (T3 and T4 respectively) with shrimp waste meal. Fish were fed with a formulated diet for 63 days. The histochemical study showed increasing in the length of villi and the number of goblet cells, and appearance necrosis in intestine layers, degeneration, shrinking of supranuclear vacuoles, detachment of base of villi, and infiltration of inflammatory eosinophilic granulocytes. Histochemical examination using alcian blue and periodic acid-Schiff revealed three types of specific goblet cells in the middle intestine which secrete neutral mucus, acidic mucus, and a mixture of acidic and neutral mucus depending on their functions. This study argued at the first time effect of formulated diet on intestine tissue histochemically. [ABSTRACT FROM AUTHOR]

We designed a marking stand for the DermojetⓇ, which substantially improves fish marking via needleless subcutaneous injection of dye. The marking stand allows to increase the nozzle-to-fish distance, adjust this position and to keep the jet injector fixed during operation as well as dye refilling. A laser pointer enables a precise and small-scale aiming. Using this marking stand we marked the caudal fin of small fish with Alcian blue for a flume experiment. In total we marked 204 gudgeon (Gobio gobio) and spirlin (Alburnoides bipunctatus) of 9–14 cm length with up to two dots per fish. Weighing, measuring and marking one sedated fish took 30 to 60 s. Immediate marking success was 100%. Fish were kept indoors in tanks for 7–12 days post-marking and the colour mark remained visible for the complete study period. During our flume experiment the colour marks at the caudal fin were detectable on all fish regardless of swimming position. With this easy and fast method fish can be marked gently, reliably and efficiently. • Application of a high-pressure jet injector for needleless and accurate colour marking of fish. • Manual for marking the caudal fin of small fish with Alcian blue.

Research-based education at the undergraduate level is ideal for fostering the training of future scientists. In an undergraduate Developmental Biology course, this learning strategy requires the availability of model species and enough research reagents, not only for technique training but also for the development of student original projects. This might be challenging in most countries, where resources are limited. Hence, there is a need to develop low-cost solutions for use in the classroom. In this study, we describe the optimization and use of two low-cost protocols in zebrafish embryos for hands-on practical sessions and project-based learning in a Developmental Biology undergraduate course in Ecuador. These protocols were designed for the practical and experimental learning of vertebrate meroblastic cleavage, gastrulation, and neural crest differentiation. The proposed protocols have been previously described in the literature and use silver nitrate and alcian blue, two relatively inexpensive reagents, to label cell membranes and cartilage. The silver nitrate protocol allows the study of cell contact formation during cleavage and the identification of cellular changes during gastrulation, including yolk internalization and epiboly. The alcian blue staining allows the analysis of cranial mesenchymal differentiation into cartilage. These protocols are ideal for practical sessions due to their ease of application, quick results, adaptability to the class schedule, and robustness in the hands of beginning researchers. Finally, these protocols are adaptable for research-based class projects.