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Context.--Distinguishing the different types of amyloid is clinically important because treatments and outcomes are different. Mass spectrometry is the new gold standard for amyloid typing, but it is costly and not widely available. Therefore, immunolabeling remains the first step in identifying the most common types of amyloidosis. In amyloid subtyping, direct immunofluorescence works well when applied to frozen sections, but immunohistochemistry on formalin-fixed, paraffin-embedded material often yields poor results, particularly for light chain amyloidosis. Recently, paraffin immunofluorescence has been described as a valuable salvage technique in renal pathology when frozen sections are not available but it has not been evaluated for extra-renal diseases. Objectives.--To evaluate the use of paraffin immunofluorescence for light-chain detection in extra-renal amyloidosis and other light-chain-associated diseases. Design.--First, we compared the staining intensity of both light chains between paraffin immunofluorescence and immunohistochemistry on a retrospective cohort of 28 cases of amyloidosis that have been previously typed. Then, we studied the role of paraffin immunofluorescence as an addition to our classical immunohistochemistry panel for amyloidosis typing. Results.--In the retrospective cohort, we found that paraffin immunofluorescence outperformed immunohistochemistry for light-chain detection. Then, in the prospective part of the study, we showed that the proportion of correctly classified cases increased from 50% to 71.9% with the adjunction of second-intention paraffin immunofluorescence to the immunohistochemistry procedure. Conclusions.--We therefore view paraffin immunofluorescence as a significant addition to the routine workflow for detection of light-chain-related diseases. (Arch Pathol Lab Med. 2021;145:343-351; doi: 10.5858/arpa.2020-0124-OA), Amyloidosis is a group or disorders associated with the presence of extracellular amyloid deposits constituted by fibrillar aggregates of misfolded protein. More than 35 protein types have been identified as [...]

An organotypic slice preparation of the adult mouse parotid salivary gland amenable to a variety of optical assessments of fluid and protein secretion dynamics is described. The semi-intact preparation rendered without the use of enzymatic treatment permitted live-cell imaging and multiphoton analysis of cellular and supracellular signals. Toward this end we demonstrated that the parotid slice is a significant addition to the repertoire of tools available to investigators to probe exocrine structure and function since there is currently no cell culture system that fully recapitulates parotid acinar cell biology. Importantly, we show that a subpopulation of the acinar cells of parotid slices can be maintained in short-term culture and retain their morphology and function for up to 2 days. This in vitro model system is a significant step forward compared with enzymatically dispersed acini that rapidly lose their morphological and functional characteristics over several hours, and it was shown to be long enough for the expression and trafficking of exogenous protein following adenoviral infection. This system is compatible with a variety of genetic and physiological approaches used to study secretory function. [Ca.sup.2+] signaling; exocytosis; immunofluorescence; electron microscopy; multiphoton; tissue culture

We have developed an immunofluorescence-based assay for high-throughput analysis of target proteins on a three-dimensional cellular microarray platform. This process integrates the use of three-dimensional cellular microarrays, which should better mimic the cellular microenvironment, with sensitive immunofluorescence detection and provides quantitative information on cell function. To demonstrate this assay platform, we examined the accumulation of the [alpha] subunit of the hypoxia-inducible factor (HIF- 1[alpha]) after chemical stimulation of human pancreatic tumor cells encapsulated in 3D alginate spots in volumes as low as 60 nL. We also tested the effect of the known dysregulator of HIF-1[alpha], 2-methoxyestradiol (2ME2), on the levels of HIF-1[alpha] using a dual microarray stamping technique. This chip-based in situ Western immunoassay protocol was able to provide quantitative information on cell function, namely, the cellular response to hypoxia mimicking conditions and the reduction of HIF-1[alpha] levels after cell treatment with 2ME2. This system is the first to enable high-content screening of cellular protein levels on a 3D human cell microarray platform.

Histone acetylation levels are regulated through the opposing activities of histone acetyltransferases (HATs) and deacetylases (HDACs). While much is known about gene-specific control of histone acetylation, little is understood about how total or cellular levels of histone acetylation are regulated. To identify regulators of cellular levels of histone acetylation, we developed an immunofluorescence-based approach to screen the single-gene deletion library of Saccharomyces cerevisiae for strains with significant reductions in cellular histone acetylation levels. Of the 4848 mutants screened, we identified 63 strains with considerable cellular hypoacetylation of N-terminal lysines in histones H3 and H4. The cellular hypoacetylation was validated for subsets of the identified strains through secondary screens including mass spectrometric analysis of individual lysines and chromatin immunoprecipitation of specific genomic loci. Among the identified mutants were several members of the Ccr4-Not complex, V-type ATPases, and vacuolar protein-sorting complexes as well as genes with unknown functions. We show that Gcn5, a major HAT in yeast, has diminished histone acetyltransferase activity in particular mutants, providing a plausible explanation for reduction of cellular acetylation levels in vivo. Our findings have revealed unexpected and novel links between histone acetylation, Gcn5 HAT activity, and diverse processes such as transcription, cellular ion homeostasis, and protein transport.

The identification and characterization of the Arabidopsis homologue of MSH4 (AtMSH4) is reported which can only be detected in floral tissues, consistent with a role in reproduction. Immunofluorescence studies indicate that its expression is limited to early meiotic prophase I, preceding the synapsis of homologous chromosomes.

Fluorescent in situ hybridization (FISH) was used assessing the state of the bacterial cell walls of the gram-positive bacterium Lactococcus lactis. FISH was utilized in studying the effect of phage infection on the capacity of L. lactis to hybridize a general HRP-labeled 16S RNA-targeted probe. The obtained results demonstrate that FISH used in conjunction with HRP-labeled probes is an excellent method in studying the cell walls of gram-positive bacteria.

To the Editor: In Vietnam, many febrile diseases such as malaria, dengue fever, Japanese encephalitis, scrub typhus, and more recently, severe acute respiratory syndrome (SARS) and avian influenza have been [...]

Byline: B. Nandeesh, Rajalakshmi. Tirumalae Background : Cutaneous vasculitis is commonly recognized and biopsied, owing to ease of access. Most biopsies are also subjected to direct immunofluorescence (DIF), though the [...]

Byline: Raghavendra. Rao, Jemima. Mellerio, Balbir. Bhogal, Richard. Groves Epidermolysis bullosa (EB) is a group of inherited, mechanobullous disorders that are caused by mutations in the structural proteins in the [...]

Byline: C. Shanmugasekar, V. Ram Ganesh, Alamelu. Jayaraman, C. Srinivas Indirect immunofluorescence with serum is used in the diagnosis of pemphigus. We report a case in whom blister fluid was [...]

* Retinoblastoma is the most common primary intraocular tumor of childhood and may be heritable or occur sporadically. Anterior diffuse retinoblastoma is an uncommon variant that is thought to be sporadic. We describe a child with anterior diffuse retinoblastoma who presented with a pseudohypopyon. Genetic analysis showed a germline mutation of the RB1 allele that is potentially heritable. Immunofluorescence staining was positive for transforming growth factor P and for vascular endothelial growth factor and negative for inducible nitric oxide synthase and for hypoxia inducible factor a in the tumor seeds, indicating acquisition of nonischemia-mediated survival factors of the tumor seeds in the aqueous humor. (Arch Pathol Lab Med. 2009;133:1215-1218), Retinoblastoma is the most common primary intraocular tumor in childhood. When a family history is present, 50% of offspring of a parent carrying an RB1 mutant allele will inherit that [...]

Byline: K. Mohan, Sathish. Pai, Raghavendra. Rao, H. Sripathi, Smitha. Prabhu Immunofluorescence is a histochemical laboratory staining technique used for demonstrating the presence of antibodies bound to antigens in tissues [...]