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The intestine is often examined histologically in connection with allergies and in search for pathological changes. To be able to examine the intestine histologically with a microscope, it must be sampled and processed correctly. For microscopic analysis, the samples have to be cut into thin sections, stained, and mounted on slides. Since it is not possible to cut fresh samples without damaging them, they must first be fixed. The most common method, which is described herein, is the fixation in formalin with subsequent embedding in paraffin and staining of the slides with hematoxylin and eosin (H&E). Hematoxylin solutions (in this case Mayer's hemalum solution) stain the acidic components of the cell, i.e., cell nuclei, blue. The staining with eosin gives a pink staining of cytoplasm. This chapter describes the method of processing intestinal tissue for paraffin-embedding, sectioning, and staining with H&E. Tissue processing can be done in tissue processing machines or manually. We describe the manual processing that is often used for smaller batches of samples.

Increasing use is being made of cell smears for cell-staining studies. Suspension cells can be attached to slides by drying, and cell smears can also be prepared from biopsy samples, such as needle aspirates, tissue scrapings, or freshly dissected tissues. In these procedures, a thin layer of cells is deposited on a dry slide by physical methods. The most important factor in obtaining good staining patterns is that the smear be only a single cell thick. Tissue smears do not preserve tissue architecture, but are useful for identifying pathological changes and infectious organisms in tissue samples. Cell smears are easily prepared and can be fixed readily by any of the methods used for attached cells., (© 2020 Cold Spring Harbor Laboratory Press.)

Precision tissue diagnostics rely on high quality input specimens so that assay results are not affected by artifact, but advances in collection and processing of tissue specimens have lagged behind innovations in diagnostic assay development. Therefore, we have designed and evaluated a novel surgical tissue collection device that maintains and monitors sample temperature and motion throughout transport so that the major preanalytical variable of tissue temperature can be controlled and measured. This device, in combination with an improved cold-hot tissue fixation protocol affords optimal biomarker preservation in less overall time, thereby simultaneously improving diagnostic quality and turnaround time. We collected 50 primary and metastatic liver tumors using a novel transport device. Tissue was fixed using a rapid cold-hot fixation protocol and immunohistochemical assays were used to assess the performance of the device, in comparison to control tissue preserved using standard clinical fixation protocol. Two pathologists evaluated the IHC studies in a blinded fashion to determine the immunophenotype of each tumor. The observed IHC staining intensities and the clinical impressions of the immunophenotypes did not differ between tissue collected with the novel device and control tissue, while improvements in processing time were achieved. The novel cold transport device and rapid fixation protocol can be successfully and safely combined and used to monitor specimen conditions, thus preserving the diagnostic utility of specimens and improving the overall turn-around time of the diagnostic process.

Phosphoproteins are the key indicators of signaling network pathway activation. Many disease treatment therapies are designed to inhibit these pathways and effective diagnostics are required to evaluate the efficacy of these treatments. Phosphoprotein IHC have been impractical for diagnostics due to inconsistent results occurring from technical limitations. We have designed and tested a novel cold transport device and rapid cold plus warm formalin fixation protocol using phosphoproteins IHC. We collected 50 liver tumors that were split into two experimental conditions: 2 + 2 rapid fixation (2 hours cold then 2 hour warm formalin) or 4 hour room-temperature formalin. We analyzed primary hepatocellular carcinoma (n = 10) and metastatic gastrointestinal tumors (n = 28) for phosphoprotein IHC markers pAKT, pERK, pSRC, pSTAT3, and pSMAD2 and compared them to slides obtained from the clinical blocks. Expression of pERK and pSRC, present in the metastatic colorectal carcinoma, were better preserved with the rapid processing protocol while pSTAT3 expression was detected in hepatocellular carcinoma. Differences in pSMAD2 expression were difficult to detect due to the ubiquitous nature of protein expression. There were only 3 cases expressing pAKT and all exhibited a dramatic loss of signal for the standard clinical workflow. The rapid cold preservation shows improvement in phosphoprotein preservation.

The osmium maceration method is a powerful technique for observing the three-dimensional ultrastructure of cellular organelles by scanning electron microscopy. In the conventional osmium maceration method, tissues are immersed in a diluted osmium tetroxide solution for several days at 20°C to remove soluble cytosolic proteins from the freeze-cracked surface of cells, and the optimal duration of this process is dependent on the cell type. To improve the efficiency of the osmium maceration procedure, we have examined systematically the relationship between the reaction temperature and time of the osmium maceration procedure. Treatment at temperatures higher than 20°C drastically shortened the time required to remove cytosolic proteins from the freeze-cracked surface of specimens with optimal durations for the osmium maceration of hepatocytes at 30, 40, 50 and 60°C being 30, 15, 5 and 1 h, respectively. Considering the stability and reproducibility of the macerated specimens, we concluded that the most appropriate temperature was 30 to 40°C. This rapid osmium maceration procedure was used successfully to observe the 3D ultrastructure of Purkinje cells in the cerebellum and proximal convoluted tubule cells in the kidney. This simple and reproducible rapid osmium maceration protocol should find wide appeal for the 3D analysis of cellular organelles in various cell types.

Histomorphology has significantly changed over the last decades due to technological achievements in immunohistochemistry (IHC) for the visualization of specific proteins and in molecular pathology, particularly in the field of in situ hybridization of small oligonucleotides and amplification of DNA and RNA amplicons. With an increased availability of suitable methods, the demands regarding the observer of histomorphological slides were the supply of complex quantitative data as well as more information about protein expression and cell-cell interactions in tissue sections. Advances in fluorescence-based multiplexed IHC techniques, such as multispectral imaging (MSI), allow the quantification of multiple proteins at the same tissue section. In histopathology, it is a well-known technique for over a decade yet harboring serious problems concerning quantitative preciseness and tissue autofluorescence of multicolor staining when using formalin-fixed, paraffin-embedded (FFPE) tissue specimen. In recent years, milestones in tissue preparation, fluorescent dyes, hardware imaging, and software analysis were achieved including automated tissue segmentation (e.g., tumor vs. stroma) as well as in cellular and subcellular multiparameter analysis.This chapter covers the role that MSI plays in anatomic pathology for the analysis of FFPE tissue sections, discusses the technical aspects of MSI, and provides a review of its application in the characterization of immune cell infiltrates and beyond regarding its prognostic and predictive value and its use for guidance of clinical decisions for immunotherapeutic strategies.

Immunohistochemistry (IHC) is a commonly used technique for protein detection in tissue sections. The method requires high-affinity antibodies that are specific for the target proteins of interest. More advanced IHC techniques have been developed to meet the need for simultaneous detection of more than one target protein in the same tissue section. This chapter provides general guidelines for double IHC staining of formalin-fixed, paraffin-embedded tissue sections. Chromogenic substrates are chosen based on their excellent contrast and compatibility with the subsequent digital image analysis.

Immunohistochemistry (IHC) is a routinely used technique in clinical diagnosis of pathological conditions and in basic and translational research. It combines anatomical, immunological, and biochemical methods and relies on the specific binding of an antibody to an antigen. Using the technique with mineralized tissues is more challenging than with soft tissues. Demineralizing the samples allows for embedding in paraffin wax, and also facilitates cryosectioning. This chapter describes methods for IHC on formaldehyde-fixed, demineralized, paraffin-embedded, or frozen sections to detect antigens in skeletal tissues.

The emerging phenomenon of cellular heterogeneity in tissue requires single-cell resolution studies. A specific challenge for suspension-based single-cell analysis is the preservation of intact cell states when single cells are isolated from tissue contexts, in order to enable downstream analyses to extract accurate, native information. We have developed DISSECT (Disaggregation for Intracellular Signaling in Single Epithelial Cells from Tissue) coupled to mass cytometry (CyTOF: Cytometry by Time-of-Flight), an experimental approach for profiling intact signaling states of single cells from epithelial tissue specimens. We have previously applied DISSECT-CyTOF to fresh mouse intestinal samples and to Formalin-Fixed, Paraffin-Embedded (FFPE) human colorectal cancer specimens. Here, we present detailed protocols for each of these procedures, as well as a new method for applying DISSECT to cryopreserved tissue slices. We present example data for using DISSECT on a cryopreserved specimen of the human colon to profile its immune and epithelial composition. These techniques can be used for high-resolution studies for monitoring disease-related alternations in different cellular compartments using specimens stored in cryopreserved or FFPE tissue banks.

Versican is a chondroitin sulfate proteoglycan found in the extracellular matrix that is important for changes in cell phenotype associated with development and disease. Versican has been shown to be involved in cardiovascular disorders, as well as lung disease and fibrosis, inflammatory bowel disease, cancer, and several other diseases that have an inflammatory component. Versican was first identified as a fibroblast proteoglycan and forms large multimolecular complexes with hyaluronan and other components of the provisional matrix during wound healing and inflammation. The biology of versican has been well studied. Versican plays a major role in embryogenesis, particularly heart formation, where versican deletion proves lethal. The ability to purify versican to characterize and to use in experimental systems is vital to defining its role in development and disease. Protein expression systems have proven challenging to obtain milligram quantities of full-length versican. Here, we describe proteoglycan biochemical purification techniques that have been developed by others, but which we have adapted to use with our source tissues and cells. We also include methods for immunohistochemical localization and quantitation of versican in tissue sections., (© 2018 Elsevier Inc. All rights reserved.)

HER2 positivity is based on the fundamental principle of amplification of the human epidermal growth factor receptor 2 (HER2) gene resulting in overexpression of the protein products . Arising from that a "HER2-positive cancer" is one that shows HER2 gene amplification and resultant protein expression as demonstrated by in situ hybridization and immunohistochemistry, respectively. Testing of the HER2 status is crucial to ensure selection of the correct patient who may benefit from target therapy for esophageal adenocarcinoma. Accurate testing is dependent on several pre-analytical and analytical factors including sample selection, laboratory techniques, and accurate interpretation of HER2 test results.

Mammalian organs comprise a variety of cells that interact with each other and have distinct biological roles. Access to evaluate and perturb intact biological systems at the cellular and molecular levels is essential to fully understand their functioning in normal and diseased conditions, yet technical limitations have constrained most research to small pieces of tissue. Tissue clearing and optogenetics can help overcome this hurdle: tissue clearing affords optical interrogation of whole organs at the molecular level, and optogenetics enables the scalable control and measurement of cellular activity with light. In this Q&A, we delineate recent advances and practical challenges associated with these two techniques when applied body-wide.

Intraoperative detection of tumorous tissue is an important unresolved issue for cancer surgery. Difficulty in differentiating between tissue types commonly results in the requirement for additional surgeries to excise unremoved cancer tissue or alternatively in the removal of excess amounts of healthy tissue. Although pathologic methods exist to determine tissue type during surgery, these methods can compromise postoperative pathology, have a lag of minutes to hours before the surgeon receives the results of the tissue analysis, and are restricted to excised tissue. In this work, we report the development of an optical fiber probe that could potentially find use as an aid for margin detection during surgery. A fluorophore-doped polymer coating is deposited on the tip of an optical fiber, which can then be used to record the pH by monitoring the emission spectra from this dye. By measuring the tissue pH and comparing with the values from regular tissue, the tissue type can be determined quickly and accurately. The use of a novel lift-and-measure technique allows for these measurements to be performed without influence from the inherent autofluorescence that commonly affects fluorescence-based measurements on biological samples. The probe developed here shows strong potential for use during surgery, as the probe design can be readily adapted to a low-cost portable configuration, which could find use in the operating theater. Use of this probe in surgery either on excised or in vivo tissue has the potential to improve success rates for complete removal of cancers. Cancer Res; 76(23); 6795-801. ©2016 AACR., (©2016 American Association for Cancer Research.)

Introduction: The objective of this work is to assess the implementation of a newly introduced medical equipment technology for the vacuum-based preservation of biological materials within an Anatomic Pathology service., Methods: The approach selected for the analysis is the Health Technology Assessment (HTA ), a comprehensive evaluation method based on relevant scientific evidence and designed to support healthcare decision makers in purchasing, replacing or disposing of technologies. The analysis focused on specific domains such as Technology, Organization, Safety and Economy., Results: The study proves that the use of such technology ensures the biological specimen to be suitably preserved (up to 72 hours), both reducing the amount of fixative being employed in the diagnostic process (30% to 55%) and resulting, in the particular context under examination, in savings of 93%., Discussion: The HTA reported no significant drawbacks related to the use of the technology being examined. Nonetheless, the workflow for managing the transfer of biological materials from the Operating Room to the Anatomic Pathology department needs to be redefined - in terms of handling, processing, storage and disposal. Other elements concerned the monitoring of storage temperature, fresh tissue handling and especially fixative amount reduction, which positively impacts on the operators' safety with regard to chemical hazards., (© Copyright Società Italiana di Anatomia Patologica e Citopatologia Diagnostica, Divisione Italiana della International Academy of Pathology.)

Identification of nematode species by using conventional methods requires fixation of the isolated material and a suitable preparation for further analyses. Tentative identification using microscopic methods should also be performed prior to initiating molecular studies. In the literature, various methods are described for the preparation of nematodes from the genus Aphelenchoides for identification and microscopic studies. The most commonly used fixatives are formalin (Timm 1969; Szczygieł & Cid del Prado Vera 1981, Crozzoli et al. 2008, Khan et al. 2008), FAA (Wasilewska 1969; Vovlas et al. 2005, Khan et al. 2007) and TAF (Hooper 1958, Chizhov et al. 2006, Jagdale & Grewal 2006).

A device to carry out all types of vacuum infiltration in a wide temperature range. The device is easy to use and allows you to simultaneously conduct the infiltration of a large number of tissue samples. The main element of the device is a temperature-controlled cuvette, the temperature of which is set in the range of 23-65 °C. The design of the cell allows pumping air from the cell to the 50-100 mm Hg. In practice, histological methods are used to remove the vacuum infiltration of air from the tissue fixation (plant tissue), to accelerate the penetration of fasteners into the tissue. To carry out such a procedure, syringes, water pump and other hand-held devicesare used. Only vacuum infiltration allows a fill lyophilized tissue in paraffin. Efficiency of the described device is checked during the experiments at various fixing plant tissue and nervous tissue filling lyophilized in paraffin.

Whole-mount in situ hybridization (WISH) is widely used to visualize the site and dynamics of gene expression during embryonic development. Various methods of probe labeling and hybridization detection are available nowadays. Meanwhile the technique was adapted to be used on many different species and has evolved from a manual to a larger scale and automated procedure. Standardized automated protocols improve the chance to compare different experimental settings reliably. The high resolution of this method is ideally suited for examination of manipulated (e.g., cloned) embryos often displaying subtle changes only. Embedding and sectioning of in situ hybridized specimen further enhance the detailed examination of their gene expression and morphology.

Procurement of fresh tissue of prostate cancer is critical for biobanking and generation of xenograft models as an important preclinical step towards new therapeutic strategies in advanced prostate cancer. However, handling of fresh radical prostatectomy specimens has been notoriously challenging given the distinctive physical properties of prostate tissue and the difficulty to identify cancer foci on gross examination. Here, we have developed a novel approach using ceramic foam plates for processing freshly cut whole mount sections from radical prostatectomy specimens without compromising further diagnostic assessment. Forty-nine radical prostatectomy specimens were processed and sectioned from the apex to the base in whole mount slices. Putative carcinoma foci were morphologically verified by frozen section analysis. The fresh whole mount slices were then laid between two ceramic foam plates and fixed overnight. To test tissue preservation after this procedure, formalin-fixed and paraffin-embedded whole mount sections were stained with hematoxylin and eosin (H&E) and analyzed by immunohistochemistry, fluorescence, and silver in situ hybridization (FISH and SISH, respectively). There were no morphological artifacts on H&E stained whole mount sections from slices that had been fixed between two plates of ceramic foam, and the histological architecture was fully retained. The quality of immunohistochemistry, FISH, and SISH was excellent. Fixing whole mount tissue slices between ceramic foam plates after frozen section examination is an excellent method for processing fresh radical prostatectomy specimens, allowing for a precise identification and collection of fresh tumor tissue without compromising further diagnostic analysis.

This article presents the best current practices for preparation of biological samples for examination as thin sections in an electron microscope. The historical development of fixation, dehydration, and embedding procedures for biological materials are reviewed for both conventional and low temperature methods. Conventional procedures for processing cells and tissues are usually done over days and often produce distortions, extractions, and other artifacts that are not acceptable for today's structural biology standards. High-pressure freezing and freeze substitution can minimize some of these artifacts. New methods that reduce the times for freeze substitution and resin embedding to a few hours are discussed as well as a new rapid room temperature method for preparing cells for on-section immunolabeling without the use of aldehyde fixatives.

Purpose: To retrospectively compare 2 methods of pre-resection, image-guided tumor localization-preoperative needle-wire localization (PNWL) and intraoperative ultrasonography-guided localization and tissue fixation (IUGLTF)-for patients with invasive breast cancer at the time of breast-conserving surgery (BCS)., Patients and Methods: We identified 118 cases in which image-guided localization was required for nonpalpable and questionably palpable tumors from a series of 204 consecutive invasive breast cancers treated by BCS. We defined a positive margin as tumor at the inked surface. We defined a close margin as tumor within 1 mm or less of the inked surface., Results: Of those 118 cases requiring pre-resection, image-guided localization, 54 patients underwent PNWL and 64 underwent IUGLTF placement. A positive margin was identified in 6 of 54 (11.1%) undergoing PNWL compared with 1 of 64 (1.6%) undergoing IUGLTF (P = .046). A positive or close margin was identified in 9 of 54 (16.7%) undergoing PNWL compared with 3 of 64 (4.7%) undergoing IUGLTF (P = .032). The mean volume and mean weight of the BCS specimens were not different in the 2 groups., Conclusion: Based on the finding of less margin positivity associated with the IUGLTF technique than the PNWL technique, we believe that the use of an IUGLTF device by surgeons during BCS could be highly advantageous in the surgical management of nonpalpable and questionably palpable invasive breast cancers., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Museum technologies provide a wide array of choice of museums to those who wish to exploit technology to attract, excite and ensure an unrivalled visitor experience, as well as capture and sustain share of mind and heart. Museum being a combination of both art and science requires skilled workmanship, meticulous planning and execution to exhibit a specimen to its optimal elegance due to its relatively smaller size and fragile nature. A well established oral pathology museum is rarely seen due to negligence of oral specimens, dearth of knowledge in this field and also available data on it. An insight on oral pathology museum, including its establishment, importance and advanced technologies to make it more simple and accessible are discussed in two parts. Part I emphasizes on basics in oral pathology museum, whereas part II highlights the specialized techniques and recent advances in museum technology. Our effort is to present this article as hands on experience for the pathologists, student population and the technicians.

Unlabelled: Formalin fixation and paraffin embedding present the standard procedures for conserving clinical tissues for histological analysis. However, molecular analysis is impaired by the cross linking properties of formalin. The PAXgene tissue system (PreAnalytix, Switzerland) is a new formalin-free tissue collection device., Aims: In this study we aimed to evaluate this new tissue preservation technique in comparison with formalin fixation and fresh frozen tissue samples., Methods: 12 melanoma biopsy samples were divided and fixed simultaneously with formalin, PAXgene or fresh frozen in liquid nitrogen and analysed with regard to morphology, immunohistochemistry,  DNA and RNA content and quality. Markers of melanocytic differentiation and tumour cell proliferation were used., Results: Morphology was well preserved in PAXPE samples. However, 5 out of 11 immunohistochemical markers showed significantly lower overall staining and staining intensity with PAXPE tissues in comparison with formalin-fixed, paraffin-embedded (FFPE). Increasing membrane permeability through adding a detergent did proportionally increase staining intensity in PAXPE samples. Amplification of different mRNA amplicons showed a direct relationship with the size of the amplicon with greater template integrity observed in PAXPE samples. Sequencing and mutational analysis of DNA samples were comparable for all the different fixation methods, while the level of DNA fragmentation seemed to be lower in PAXPE compared with FFPE tissues., Conclusions: The switch from formalin to PAXgene fixation would require a re-evaluation of immunohistochemical markers and staining procedures originally developed for FFPE tissues. Our data demonstrate that PAXPE fixation offers some advantages concerning molecular analysis. However, these advantages would not justify substituting formalin fixation in any routine pathology laboratory.

The purpose of this protocol is to take any biopsy or whole organ tissue from animals or human, formalin-fix the specimen to preserve the current state of the tissue, and embed it into a paraffin block and for future immunohistochemistry experiments (If you intend to fix cells, check the alternative protocols: Preparation of Cells for Microscopy using Cytospin, Preparation of Cells for Microscopy using Chamber Slides and Coverslips, or Preparation of Cells for Microscopy using 'Cell Blocks')., (© 2013 Elsevier Inc. All rights reserved.)

A pre-cryogenic holder (cryo-holder) facilitating cryo-specimen observation under a conventional scanning electron microscope (SEM) is described. This cryo-holder includes a specimen-holding unit (the stub) and a cryogenic energy-storing unit (a composite of three cylinders assembled with a screw). After cooling, the cryo-holder can continue supplying cryogenic energy to extend the observation time for the specimen in a conventional SEM. Moreover, the cryogenic energy-storing unit could retain appropriate liquid nitrogen that can evaporate to prevent frost deposition on the surface of the specimen. This device is proved feasible for various tissues and cells, and can be applied to the fields of both biology and material science. We have employed this novel cryo-holder for observation of yeast cells, trichome, and epidermal cells in the leaf of Arabidopsis thaliana, compound eyes of insects, red blood cells, filiform papillae on the surface of rat tongue, agar medium, water molecules, penicillium, etc. All results suggested that the newly designed cryo-holder is applicable for cryo-specimen observation under a conventional SEM without cooling system. Most importantly, the design of this cryo-holder is simple and easy to operate and could adapt a conventional SEM to a plain type cryo-SEM affordable for most laboratories., (Copyright © 2011 Wiley Periodicals, Inc.)

Background: Stereology is an essential method for quantitative analysis of lung structure. Adequate fixation is a prerequisite for stereological analysis to avoid bias in pulmonary tissue, dimensions and structural details. We present a technique for in situ fixation of large animal lungs for stereological analysis, based on closed loop perfusion fixation., Materials and Methods: Twenty anesthetized ventilated pigs (30 ± 3 kg) underwent cannulation of the pulmonary artery and ligation of the right hilus. Following circulatory arrest a continuous positive pressure of 12 mbar was applied to the airways and lung perfusion started with the fixative solution (1.5% paraformaldehyde; 1.5% glutaraldehyde in 0.15 M HEPES). In five animals, a single-pass perfusion technique was performed, in 15 subsequent animals, the closed-loop technique was applied. Afterwards, lungs were removed, externally postfixed in the recycled fixative solution, and stored at 4 °C. Fifteen lung specimens underwent stereological analysis with volume estimation and subsequent systematic uniform random sampling for light and electron microscopic analysis., Results: Singlepass perfusion did not result in satisfactory fixation. Left lung closed loop perfusion rate was 0.5-0.7 L/min with total median [min-max] perfusion time of 15 min (11-19). Perfusion pressure was 15 mm Hg (9-33). Subsequent lung analysis revealed well-preserved cell and tissue ultrastructure., Conclusion: The closed loop perfusion technique represents a valuable and reproducible fixation method in large animal models. Pressure controlled fixation perfusion results in high-quality preservation of in situ parenchymal architecture of lungs with or without injury, which is ideally suited for quantitative assessment of lung structure by stereology., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Resonance Raman microscopy is well suited to examine living bacterial samples without further preparation. Therefore, comparatively little thought has been given to its compatibility with common fixation methods. However, fixation of cell samples is a very important tool in the microbiological sciences, allowing the preservation of samples in a specific condition for further examination, future measurements, transport, or later reference. We examined the effects of three common fixatives-ethanol, formaldehyde solution, and gentle heat--on the resonant Raman spectrum of three generic bacteria species, Rhodobacter sphaeroides DSM 158(T), Rhodopseudomonas palustris DSM 123(T), and Rhodospirillum rubrum DSM 467(T), holding carotenoid- and heme-chromophores in confocal Raman microscopy. In addition, we analyzed the effect of poly-L-lysine coating of microscope slides, widely used for mounting biological and medical samples, on subsequent confocal Raman measurements of native and fixed samples. The results indicate that ethanol is preferable to formaldehyde as fixative if applied for less than 24 h, whereas heat fixation has a strong, detrimental effect on the resonant Raman spectrum of bacteria. Formaldehyde fixation excels at fixation times above 24 h, but causes an overall reduction in signal intensity. Poly-L-lysine coating has no discernable effect on the Raman spectra of samples fixed with ethanol or heat, but it further decreases the signal intensity, especially at higher wavenumbers, in the spectra of samples fixed with formaldehyde., (Copyright © 2010 Wiley-Liss, Inc.)

Activating transcription factor 3 (ATF3) gene encodes a member of the ATF family of transcription factors and is induced by various stress signals, including many of those that induce the unfolded protein response (UPR). Emerging evidence suggests that ATF3 is a hub of the cellular adaptive-response network and studies using various mouse models indicate that ATF3 plays a role in the pathogenesis of various diseases. One way to investigate the potential relevance of ATF3 to human diseases is to determine its expression in patient samples and test whether it correlates with disease progression or clinical outcomes. Due to the scarcity and preciousness of patient samples, methods that can detect ATF3 on archival tissue sections would greatly facilitate this research. In this chapter, we briefly review the roles of ATF3 in cellular adaptive-response and UPR, and then describe the detailed steps and tips that we developed based on general immunohistochemistry (IHC) protocols to detect ATF3 on paraffin embedded sections., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The authors describe a simple method for making formalin or isopropyl alcohol vapour fixed cell blocks from fine needle aspiration cytology specimens that we refer to as 'The Poor Man's Cell Block.'

Introduction: Vocal cords paralysis is the second most frequent cause of laryngeal stridor in children. Symptoms of congenital vocal cords paralysis can occur shortly after birth or later. Vocal cords paralysis can be unilateral or bilateral. Symptoms of unilateral paralysis include hoarse weeping or stridor during a deep inhalation. In children unilateral vocal cords paralysis often retreats spontaneously or can be completely compensated. Children with bilateral vocal cords paralysis present mainly breathing disorders while phonation is normal. Symptoms are different, starting from complete occlusion of respiratory tracts and ending on small symptoms connected with the lack of effort tolerance. When symptoms are severe, patients from this group require a tracheotomy. The lack of restoration of normal function of vocal cords or lack of complete compensation and maintenance of symptoms are an indication for surgical treatment., Objective: The aim of this study is to present results of the treatment of bilateral vocal cords paralysis in children using the endoscopic method of laterofixation of vocal cords., Material and Methods: In the Pediatric ENT Department between 1998 and 2009 sixty four children with dyspnoea and/or phonation disorders caused by vocal cords paralysis were treated., Results: In ten cases laterofixation of vocal cords was performed, in most cases with good result. In this article the authors present the method of endoscopic laterofixation and achieved results., Conclusions: Endoscopic laterofixation of vocal cords in children is a safe and an easy method of surgical treatment of bilateral vocal cords paralysis. This method can be used as a first and often as a one stage treatment of vocal cords paralysis. In some cases this procedure is insufficient and has to be completed with other methods., (Copyright (c) 2010 Elsevier Ireland Ltd. All rights reserved.)

Autophagy is a compensatory pathway involving isolation and subsequent degradation of cytosolic material and organelles in eukaryotic cells.(1) The autophagic process can provide a "housekeeping" function by removing damaged proteins and organelles in a selective or nonselective fashion in order to exert a protective effect following stress.(2) Remarkably, after being discovered to be much more of a targeted process than a random one, the role of autophagy became implicated in many normal cellular and disease processes.(3) Several methodologies are routinely employed to monitor the entire autophagic process.(4) Microtubule-associated protein light chain 3, a mammalian homolog of yeast Atg8, has been widely used as a specific marker to monitor autophagy in numerous cell types.(5) While monitoring autophagic flux is extremely important, it is also beneficial to perform a detailed analysis by electron microscopy (EM) to evaluate changes in various autophagic structures, quantify the areas involved, and determine if any particular organelle(s) or area of the cell cytoplasm is being targeted for degradation.(6) The following article describes methods to localize and quantify subcellular areas of autophagy using transmission EM. Also discussed are methods for subcellular localization of specific proteins by employing immunogold EM; this method becomes particularly useful in detecting early changes in cellular homeostasis that may occur before later signs of cellular insult can be observed morphologically.

High-pressure freezing (HPF) has been around since the mid-1980s as a cryopreparation technique for biological electron microscopy. It has taken quite some time to "catch on" but with the recent interest in cellular tomography and electron microscopy of vitreous cryosections it has been used more frequently. While HPF is relatively easy to do, there are a number of steps, such as loading the sample into the specimen carrier correctly, that are critical to the success of this method. In this chapter we discuss some of the "little" things that can make the difference between successful or unsuccessful freezing. We cover all aspects of HPF, from specimen loading to removing your sample from the carriers in polymerized resin. Our goal is to make it easier and more reliable for HPF users to get well-frozen samples for their research., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Background: An anterolateral approach to the tibial tunnel of posterior cruciate ligament reconstruction is used to reduce the sharpness of the graft-tunnel angle, the so-called killer turn effect. However, with the anterolateral approach, the tunnel might be widened into an ovoid shape because of the small angle between the tunnel and the anterolateral cortex., Hypothesis: The fixation strength of the posterior cruciate ligament graft in the tibial tunnel will be weaker in the anterolateral approach compared with the anteromedial approach., Study Design: Controlled laboratory study., Methods: Twenty paired cadaveric tibias were used. Tibial tunnels were made using following approaches: an anteromedial approach for 10 tibias and an anterolateral approach for 10 tibias. The anterior cortex-tunnel angle and the diameter of the tunnel entrance were measured by 2-dimensional computed tomographic scans. After fixation of the Achilles tendon allograft with a biodegradable screw, the maximal strength of the graft at failure was measured using a materials testing machine., Results: The mean cortex-tunnel angle was 47.5 degrees +/- 9.3 degrees in the anteromedial approach group and 28.3 degrees +/- 7.4 degrees in the anterolateral approach group. The mean long diameter of the tunnels in the anteromedial approach group was 10.6 +/- 1.0 mm and in the anterolateral approach group it was 14.0 +/- 1.5 mm. These two parameters showed statistically significant differences between the 2 groups (P < .01). The mean maximum load at failure for the anteromedial approach group was 385.4 +/- 139.7 N, and for the anterolateral approach group it was 225.1 +/- 144.1 N. This difference was statistically significant (P = .021)., Conclusion: The anterolateral approach resulted in a tunnel with a wider entrance, a more acute cortex-tunnel angle, and a lower maximal load at failure compared with tunnels created using the anteromedial approach., Clinical Relevance: The use of additional fixation methods, such as post ties or ligament washers and screws, should be considered when using an anterolateral approach for tibial tunnel of posterior cruciate ligament reconstruction.

The masking effects of antigens by chemical fixation, processing, embedding media interactions, represent a serious problem for immunohistochemical purposes. Fortunately, different approaches in antigen retrieval exist. These techniques are relatively recent and continuously expanding. This review focuses on the present state of the art in antigen retrieval methods for immunohistochemistry in light and electron microscopy. Moreover, a brief discussion on the chemical aspects of fixation, mechanism of retrieval, as well as its efficacy, is given.

High-performance liquid chromatography with tandem mass spectrometry detection (LC-MS/MS) allows a highly selective, sensitive, simultaneous analysis for prostanoids (PG) without derivatization. However, high chemical background noise reduces LC-MS/MS selectivity and sensitivity for brain PG analysis. Four common methods using different solvent systems for PG extraction were tested. Although these methods had the same recovery of PG, the modified acetone extraction followed by liquid/liquid purification had the greatest sensitivity. This method combined with hexane/2-propanol extraction permits the simultaneous analysis of other lipid molecules and PG in the same extract. We also determined that PG mass in brain powder stored at -80 degrees C was reduced 2- to 4- fold in 4 weeks; however, PG were stable for long periods (>3 months) in hexane/2-propanol extracts. PG mass was increased significantly when mice were euthanized by decapitation and the brains rapidly flash-frozen rather than euthanized using head-focused microwave irradiation. This reduction is not the result of PG trapping or destruction in microwave-irradiated brains, demonstrating its importance in limiting mass artifacts during brain PG analysis. Our improved procedure for brain PG analysis provides a reliable, rapid means to detect changes in brain PG mass under both basal and pathological conditions and demonstrates the importance of sample preparation in this process.

Patterning of zebrafish and other vertebrate embryos proceeds according to consistent, predictable developmental time courses. Because zebrafish spawn primarily during the first few hours after dawn, many important developmental stages typically occur during the middle of the night. As an automatic, accurate way to fix embryos at these inconvenient times, we have developed the Time Reaper 5-Channel Automatic Liquid Dispenser (TimeR). The TimeR delivers up to 50 mL of liquid to embryos in a Petri dish at preset times. We have used the TimeR to deliver paraformaldehyde and fix zebrafish embryos at different stages of development. We find that the pattern of expression for a number of genes is indistinguishable between embryos fixed manually and with the TimeR. The TimeR is also suitable for fixing embryos for whole-mount immunostaining, but care needs to be taken to find conditions that preserve the antibody's epitope. The TimeR is inexpensive to make, and can be constructed using tools present in most machine shops. In addition to fixing embryos, the TimeR will be useful for any experiment that requires automatic delivery of milliliter amounts of liquid.

Periorbital rejuvenation can enhance a patient's appearance, with changes of only a few millimeters making a significant impact. Many patients undergoing blepharoplasty often have mild brow or midfacial changes for which they are unwilling to undergo additional concurrent forehead or midface procedures, however, because these procedures may be associated with an unacceptable postoperative recovery and may not provide adequate tissue fixation. This article describes limited incision procedures capable of providing excellent elevation and support to the brow and midface that can be incorporated easily into a comprehensive periorbital rejuvenation treatment plan using semi-permanent subperiosteal fixation devices.

Mechanical testing of soft musculoskeletal structures like tendons and ligaments are essential to medical advances. A long-standing limitation for testing these structures in isolation has been the ability to solidly fix both ends of the tendon. Cryogenic fixation technology was leveraged into the development of a dual cryogenic fixation (DCF) device. Results of the study show that the DCF allows tendons to be tested in isolation, at physiologic temperatures, with excellent reproducibility.

Formalin fixation and paraffin embedding are conventional tissue preservation and processing methods used for histologic diagnosis in over 90% of cases. However, formalin fixation has three disadvantages: (1) slow fixation (16-24 h) hinders intraoperative decision making, (2) slow quenching of enzymatic activity causes RNA degradation, and (3) extensive molecule modification affects protein antigenicity. Applying high-frequency, high-intensity ultrasound to the formalin fixative cuts fixation time to 5-15 min. Fixation of various tissues such as lymph node, brain, breast, and prostate suggests that, compared to the conventional method, implementation of ultrasound retains superior and more uniform tissue morphology preservation. Less protein antigenicity is altered so that rapid immunohistochemical reactions occur with higher sensitivity and intensity, reducing the need for antigen retrieval pretreatment. Better RNA preservation results in stronger signals in in situ hybridization and longer RNA fragments extracted from fixed tissues, probably due to rapid inhibition of endogenous RNase activity. Molecules extracted from ultrasound-fixed tissues are of greater integrity and quantity compared to conventionally fixed tissues, and thus better support downstream molecular analyses. Overall, ultrasound-facilitated tissue preservation can provide rapid and improved morphological and molecular preservation to better accommodate both traditional and molecular diagnoses.

Aims: To examine the impact of microwave (MW) tissue processing on turnaround times (TATs) in a routine diagnostic laboratory., Methods: A retrospective review of TATs for tissue processing (specimen receipt to completion of H&E-stained section) and pathologists' report generation (receipt of stained section to validation of completed report) for small biopsies processed in a MW-histoprocessor (Milestone RHS-2, Italy) was performed and compared with similar TATs for specimens processed conventionally prior to the introduction of MW histoprocessing., Results and Conclusions: The TAT for conventional tissue processing was almost 21 h compared with 6.5 h by MW-processing. Reporting TATs fell from 4.3 to 3.2 hours per case, respectively, largely because stained sections became available for reporting in the early afternoon instead of towards the end of the working day. If small biopsies are triaged and handled separately, the TATs can be further reduced, and in selected urgent cases, sections can be available for diagnosis within 90 minutes. The quality of MW processed sections was indistinguishable from those obtained with routine 4-hour processing. The drastic reduction in TATs signifies a new era in histopathological diagnosis in which the majority of reports can be generated within 24 hours of specimen receipt with attendant impact on patient management and hospitalisation costs.

We studied the effect of a fully automated microwave-assisted rapid tissue processor (RTP) on histologic examination and on the turnaround time for surgical pathology reports. A quality assurance program reviewed the histologic sections obtained by the rapid processing method for the last 3 calendar years. In addition, the histologic results from this method were compared blindly with those obtained from the conventional overnight tissue processing (CTP) method by 9 pathologists with different levels of experience. The surgical pathology turnaround times for 1 year of use of the RTP were compared with the last year for CTP. We found that the RTP reproducibly yielded histologic material comparable in quality to CTP. The turnaround time for surgical pathology reports was improved substantially, and, in particular, same-day reporting was achieved in approximately 55% of cases compared with fewer than 1% before use of the RTP. Moreover, use of the RTP enhanced safety by eliminating formalin and xylene from the procedure.

The current strategy for the control of tuberculosis (TB) relies on early diagnosis, and smear microscopy is an essential component of the laboratory diagnosis of TB in most countries with a high prevalence of the disease. However, even simple smear microscopy examination is far from satisfactory because staining results can vary among individual technicians. In an effort to minimize variations in manual staining procedures, we developed an automated stainer for AFB and evaluated its usefulness in comparison with manual staining. The key feature of our automated stainer is a heating apparatus required for fixation and carbol-fuchsin staining. After smear slides are placed into the machine, the entire staining process is fully automated, from fixation to final washing and drying. With the automated methods, five slides can be fixed and stained in 21 min at consistent high quality. Using sputum samples from 91 TB patients, the staining results of the automated stainer were compared blindly with those of manual staining. The concordance rate between the two methods was 94.5%. In addition, there was no significant difference in the rate of detection of AFB in the sputum samples. Although further optimization of the auto staining procedures is required, the results indicate that the automated AFB stainer developed in this study looks promising for use in clinical mycobacteriology laboratory in order to minimize personal variation during AFB staining.

We performed a new technique for foetal brain fixation and extraction that offers a remarkable reduction in sampling time facilitates specimen handling while conserving high quality. With the new fixation method, it is possible to obtain samples adequate for macroscopic and microscopic observations and immunohistochemical analysis. The technique involves the creation of an ex vacuo phenomenon in the subarachnoideal space prior to injecting the fixative solution. In this manner, the solution is homogenously distributed. Ease, reproducibility and the possibility of standardizing the procedure are the principal advantages. Low costs, reduced wording time and less need for human resources are other advantages. Histologically, we obtained, quickly, high quality slides with routine and immunohistochemical stains. Disadvantages of the technique derive from the use of formaldehyde and glacial acetic acid, rather than water to wash the samples; thus one must work in a well ventilated area, with gloves, protective glasses and an adequate lab coat to avoid skin and respiratory tract damage.

Background: Healing complications following median sternotomy commonly include instability, nonunion, and infection. They are associated with a high mortality rate if mediastinitis supervenes. Closure complications are best avoided by improving stability at the union, but there has thus far been no widespread agreement among surgeons about relative superiority among the available closure techniques., Materials and Methods: A biological sternotomy closure model was developed utilizing whole porcine sterna. A special stainless-steel clamp with multiple spikes was created to reliably attach the sterna to a biomechanical testing device., Results: Two wiring techniques, single peristernal and pericostal figure-eight, were used in 14 fresh cadaveric porcine sterna. The more rigid closure utilized single peristernal wires (P < 0.0001). There was no tissue associated with clamp spikes penetrating the specimen's layers, and there was no clamp displacement even at closure failure loads., Conclusions: The porcine sternotomy model is a valuable tool for comparing closure techniques based on geometrical and mechanical wiring patterns. The model's low cost and easy reproducibility make it a promising first step in sternotomy closure research. The stainless-steel clamp used in the porcine model provided reliable repeat specimen fixation.