A Standardized and Reproducible Workflow for Membrane Glass Slides in Routine Histology and Spatial Proteomics.

Defining the molecular phenotype of single cells in situ is key for understanding tissue architecture in health and disease. Advanced imaging platforms have recently been joined by spatial omics technologies, promising unparalleled insights into the molecular landscape of biological samples. Furthermore, high-precision laser microdissection (LMD) of tissue on membrane glass slides is a powerful method for spatial omics technologies and single-cell type spatial proteomics in particular. However, current histology protocols have not been compatible with glass membrane slides and LMD for automated staining platforms and routine histology procedures. This has prevented the combination of advanced staining procedures with LMD. In this study, we describe a novel method for handling glass membrane slides that enables automated eight-color multiplexed immunofluorescence staining and high-quality imaging followed by precise laser-guided extraction of single cells. The key advance is the glycerol-based modification of heat-induced epitope retrieval protocols, termed "G-HIER." We find that this altered antigen-retrieval solution prevents membrane distortion. Importantly, G-HIER is fully compatible with current antigen retrieval workflows and mass spectrometry-based proteomics and does not affect proteome depth or quality. To demonstrate the versatility of G-HIER for spatial proteomics, we apply the recently introduced deep visual proteomics technology to perform single-cell type analysis of adjacent suprabasal and basal keratinocytes of human skin. G-HIER overcomes previous incompatibility of standard and advanced staining protocols with membrane glass slides and enables robust integration with routine histology procedures, high-throughput multiplexed imaging, and sophisticated downstream spatial omics technologies.

Methods to study adult hippocampal neurogenesis in humans and across the phylogeny.

The hippocampus hosts the continuous addition of new neurons throughout life-a phenomenon named adult hippocampal neurogenesis (AHN). Here we revisit the occurrence of AHN in more than 110 mammalian species, including humans, and discuss the further validation of these data by single-cell RNAseq and other alternative techniques. In this regard, our recent studies have addressed the long-standing controversy in the field, namely whether cells positive for AHN markers are present in the adult human dentate gyrus (DG). Here we review how we developed a tightly controlled methodology, based on the use of high-quality brain samples (characterized by short postmortem delays and ≤24 h of fixation in freshly prepared 4% paraformaldehyde), to address human AHN. We review that the detection of AHN markers in samples fixed for 24 h required mild antigen retrieval and chemical elimination of autofluorescence. However, these steps were not necessary for samples subjected to shorter fixation periods. Moreover, the detection of labile epitopes (such as Nestin) in the human hippocampus required the use of mild detergents. The application of this strictly controlled methodology allowed reconstruction of the entire AHN process, thus revealing the presence of neural stem cells, proliferative progenitors, neuroblasts, and immature neurons at distinct stages of differentiation in the human DG. The data reviewed here demonstrate that methodology is of utmost importance when studying AHN by means of distinct techniques across the phylogenetic scale. In this regard, we summarize the major findings made by our group that emphasize that overlooking fundamental technical principles might have consequences for any given research field.

Impact of antigen retrieval protocols on the immunohistochemical detection of epigenetic DNA modifications.

This study compares three different pretreatment protocols for the immunohistochemical detection of 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) in nuclear DNA. The human biological samples analyzed included formalin-fixed and paraffin-embedded (FFPE) normal squamous epithelium, ethanol-fixed cultured cells, and metaphase chromosomes. The antigen retrieval methods included low pH Citrate and high pH Tris-ethylenediaminetetraacetic acid (EDTA) protocols, as well as a method using Pepsin pretreatment combined with HCl for DNA denaturation. A gradual increase in the detection levels of 5-mC and 5-hmC was observed when going from Citrate via Tris/EDTA to Pepsin/HCl retrieval. While the Citrate retrieval protocol was the least efficient for the detection of 5-mC and 5-hmC, it did preserve nuclear morphology and enabled visualization of differences in intra- and internuclear distribution patterns in tissue and cell culture samples by single- and double-fluorescence detection. Quantification of (hydroxy)methylation levels in FFPE material demonstrated a significant heterogeneity and differences in 5-mC and 5-hmC levels within and between nuclei in the different compartments of normal squamous epithelium. It was concluded that immunohistochemical detection of 5-mC and 5-hmC enables the correlation of these DNA modifications with histomorphological features in heterogeneous tissues, but this is influenced by different pretreatment protocols that must be carefully chosen to allow an appropriate interpretation of these epigenetic switches.

Systematic evaluation and optimization of protein extraction parameters in diagnostic FFPE specimens.

OBJECTIVES: Formalin-fixed paraffin-embedded (FFPE) tissue is the standard material for diagnostic pathology but poses relevant hurdles to accurate protein extraction due to cross-linking and chemical alterations. While numerous extraction protocols and chemicals have been described, systematic comparative analyses are limited. Various parameters were thus investigated in their qualitative and quantitative effects on protein extraction (PE) efficacy. Special emphasis was put on preservation of membrane proteins (MP) as key subgroup of functionally relevant proteins. METHODS: Using the example of urothelial carcinoma, FFPE tissue sections were subjected to various deparaffinization, protein extraction and antigen retrieval protocols and buffers as well as different extraction techniques. Performance was measured by protein concentration and western blot analysis of cellular compartment markers as well as liquid chromatography-coupled mass spectrometry (LC-MS). RESULTS: Commercially available extraction buffers showed reduced extraction of MPs and came at considerably increased costs. On-slide extraction did not improve PE whereas several other preanalytical steps could be simplified. Systematic variation of temperature and exposure duration demonstrated a quantitatively relevant corridor of optimal antigen retrieval. CONCLUSIONS: Preanalytical protein extraction can be optimized at various levels to improve unbiased protein extraction and to reduce time and costs.

Methods for restoration of ki67 antigenicity in aged paraffin tissue blocks.

Pathology archives are a treasure trove of paraffin embedded tissue spanning many years and covering a wide variety of tissues and diseases. The possibility of using old archival formalin fixed paraffin embedded (FFPE) tissues for diagnostic updates and research projects is a widespread need and it requires archives of stable, well-preserved samples. Immunohistochemistry performed on old archival paraffin blocks may give unreliable results, in particular for some antigens, such as Ki67. In consideration of this phenomenon, our aim is to comprehensively test and identify methods which may be used to obtain Ki67 immunohistochemical reactions of good quality from old archival FFPE blocks. Various methods were tested in order to evaluate their possible efficacy in increasing Ki67 immunointensity in a collection of 40-year-old, archival blocks including re-embedding, with deeper sectioning of tissue from the block and increasing heat-based pretreatment times (20 cases) and re-processing (20 cases). All reactions were performed using an automated immunostainer and Ki67 stained immunosections compared using a visual colour-based scale (the first immunostained section was considered as baseline). The combination of deep sectioning (1000 µM) and prolonged heat-based pretreatment (64 min) markedly increased immunoreactivity for Ki67. Re-embedding and reprocessing did not have a significant effect. Large tissue samples showed heterogeneity of Ki67 immunoexpression between the periphery of the sample and the central area. In conclusion, the study defines a useful protocol to increase antigen retrieval applicable to dated archival tissues.

Fit-for-purpose based testing and validation of antibodies to amino- and carboxy-terminal domains of cannabinoid receptor 1.

Specific and selective anti-CB1 antibodies are among the most powerful research tools to unravel the complex biological processes mediated by the CB1 receptor in both physiological and pathological conditions. However, low performance of antibodies remains a major source of inconsistency between results from different laboratories. Using a variety of techniques, including some of the most commonly accepted ones for antibody specificity testing, we identified three of five commercial antibodies against different regions of CB1 receptor as the best choice for specific end-use purposes. Specifically, an antibody against a long fragment of the extracellular amino tail of CB1 receptor (but not one against a short sequence of the extreme amino-terminus) detected strong surface staining when applied to live cells, whereas two different antibodies against an identical fragment of the extreme carboxy-terminus of CB1 receptor (but not one against an upstream peptide) showed acceptable performance on all platforms, although they behaved differently in immunohistochemical assays depending on the tissue fixation procedure used and showed different specificity in Western blot assays, which made each of them particularly suitable for one of those techniques. Our results provide a framework to interpret past and future results derived from the use of different anti-CB1 antibodies in the context of current knowledge about the CB1 receptor at the molecular level, and highlight the need for an adequate validation for specific purposes, not only before antibodies are placed on the market, but also before the decision to discontinue them is made.

Automated Quantitative Image Evaluation of Antigen Retrieval Methods for 17 Antibodies in Placentation and Implantation Diagnostic and Research.

Immunostaining in clinical routine and research highly depends on standardized staining methods and quantitative image analyses. We qualitatively and quantitatively compared antigen retrieval methods (no pretreatment, pretreatment with pepsin, and heat-induced pretreatment with pH 6 or pH 9) for 17 antibodies relevant for placenta and implantation diagnostics and research. Using our newly established, comprehensive automated quantitative image analysis approach, fluorescent signal intensities were evaluated. Automated quantitative image analysis found that 9 out of 17 antibodies needed antigen retrieval to show positive staining. Heat induction proved to be the most efficient form of antigen retrieval. Eight markers stained positive after pepsin digestion, with ß-hCG and vWF showing enhanced staining intensities. To avoid the misinterpretation of quantitative image data, the qualitative aspect should always be considered. Results from native placental tissue were compared with sections of a placental invasion model based on thermo-sensitive scaffolds. Immunostaining on placentas in vitro leads to new insights into fetal development and maternal pathophysiological pathways, as pregnant women are justifiably excluded from clinical studies. Thus, there is a clear need for the assessment of reliable immunofluorescent staining and pretreatment methods. Our evaluation offers a powerful tool for antibody and pretreatment selection in placental research providing objective and precise results.

MGMT immunohistochemistry in pituitary tumors: controversies with clinical implications.

INTRODUCTION: Temozolomide (TMZ) is currently considered as a rational therapeutic option for patients with progressively aggressive pituitary adenomas and carcinomas not responding to conventional therapies. Administration of TMZ results in clinical response and improvement in survival of many of these patients depending upon the expression of the DNA repair enzyme O-6 methylguanine DNA transferase (MGMT). Low or negative MGMT immunoreactivity predicts responsiveness to TMZ therapy. Therefore, MGMT serves as a criterion to select candidate patients anticipating response to treatment. MATERIALS AND METHODS: The MGMT expression was investigated in 25 pituitary adenomas with Ki-67 labeling index more that 3% and p53 expression, using various antigen retrieval protocols. After direct application of the antibody, only one adenoma yielded positive for MGMT. However, after pretreatment of tissue sections with antigen retrieval protocols, another 3 adenomas, initially negative turned to positive. CONCLUSIONS: These findings could explain lack of response to TMZ treatment in patients with false negative MGMT immunohistochemistry. Evaluation of tumor samples for MGMT expression should carefully be carried-out using the optimum immunohistochemical protocol to obtain consistent and reliable results that help to identify patients that could respond to TMZ therapy.

Re-evaluation of MIB-1 immunostaining for diagnosing hyalinizing trabecular tumour of the thyroid: semi-automated techniques with manual antigen retrieval are more accurate than fully automated techniques.

Hyalinizing trabecular tumour (HTT) immunohistochemically shows cell membranous immunoreactivity for MIB-1. This aberrant immunoreactivity is an important factor for the diagnosis of HTT. However, fully automated stainers frequently fail to confirm the immunoreactivity. The aim of this study is to investigate the cause of false negative cell membranous immunoreactivity for MIB-1 in HTT using fully automated stainers, to determine potential reasons for the problem, and to establish methods confirming cell membranous immunoreactivity for MIB-1 in HTT. Six participating institutions examined immunoreactivity for MIB-1 in 10 HTT cases using two approaches: fully automated and semi-automated methods. In the latter, antigen retrieval was carried out using manual methods adopted for routine assays at each institute. The autostainers used included the BOND-MAX, BOND-III, Benchmark XT, and Omnis systems. Using fully automated methods, institute E showed cell membranous MIB-1 positivity in all HTT cases. In contrast, at institute D, all HTT cases were negative. The positive rates of the remaining four institutes ranged from 10% to 20%. The incidence of positive cases using semi-automated methods was 100%, 90%, 90%, 30%, 80%, and 100% at institutes A, B, C, D, E, and F, respectively. We assert that antigen retrieval should be conducted manually for diagnosis of HTT; furthermore, definitively diagnosed HTT should be prepared as the external positive control.

Antigen retrieval prior to on-tissue digestion of formalin-fixed paraffin-embedded tumour tissue sections yields oxidation of proline residues.

MALDI-mass spectrometry imaging (MALDI-MSI) has been shown to allow the study of protein distribution and identification directly within formalin-fixed paraffin-embedded (FFPE) tissue sections. However, direct protein identification from tissue sections remains challenging due to signal interferences and/or existing post-translational or other chemical modifications. The use of antigen retrieval (AR) has been demonstrated for unlocking proteins prior to in situ enzymatic digestion and MALDI-MSI analysis of FFPE tissue sections. In the work reported here, the identification of proline oxidation, which may occur when performing the AR protocol, is described. This facilitated and considerably increased the number of identified peptides when adding proline oxidation as a variable modification to the MASCOT search criteria. This article is part of a Special Issue entitled: MALDI Imaging, edited by Dr. Corinna Henkel and Prof. Peter Hoffmann.

A microwave antigen retrieval method using two heating steps for enhanced immunostaining on aldehyde-fixed paraffin-embedded tissue sections.

Antigen retrieval is an immunohistochemical procedure that results in better exposure of target antigens in aldehyde-fixed, paraffin-embedded tissue sections to antibodies. However, the commercially recommended or conventional protocols for antigen retrieval do not always succeed in expressing the target antigen. Here, an improved method was developed for antigen retrieval from aldehyde-fixed, paraffin-embedded histological sections. Proliferating cell nuclear antigen (PCNA), tight junction proteins Claudin-2 and Claudin-7, and water channel aquaporins in kidney tissue were selected as test antigens. Typically, PCNA and Claudin-2 and Claudin-7 show negative, weak, or nonspecific immunoreactions with conventional antigen retrieval methods using microwave heating. In the present study, microwave heating was performed twice with an interval of 30 min between the two steps to allow the buffer solution to cool. Sodium citrate buffer (10 mM sodium citrate, pH 6.0) was used for PCNA, and Tris-EDTA buffer (10 mM Tris, 1 mM EDTA, pH 9.0) was used for the Claudins. Compared with conventionally prepared tissues, the tissues exhibited both enhanced and specific immunostaining, and well-preserved morphology. In conclusion, the conventional protocol could be supplemented with a second microwave heating step to improve the expression of antigens that do not respond well to the conventional method.

Immunohistochemical techniques for the human inner ear.

In this review, we provide a description of the recent methods used for immunohistochemical staining of the human inner ear using formalin-fixed frozen, paraffin and celloidin-embedded sections. We also show the application of these immunohistochemical methods in auditory and vestibular endorgans microdissected from the human temporal bone. We compare the advantages and disadvantages of immunohistochemistry (IHC) in the different types of embedding media. IHC in frozen and paraffin-embedded sections yields a robust immunoreactive signal. Both frozen and paraffin sections would be the best alternative in the case where celloidin-embedding technique is not available. IHC in whole endorgans yields excellent results and can be used when desiring to detect regional variations of protein expression in the sensory epithelia. One advantage of microdissection is that the tissue is processed immediately and IHC can be made within 1 week of temporal bone collection. A second advantage of microdissection is the excellent preservation of both morphology and antigenicity. Using celloidin-embedded inner ear sections, we were able to detect several antigens by IHC and immunofluorescence using antigen retrieval methods. These techniques, previously applied only in animal models, allow for the study of numerous important proteins expressed in the human temporal bone potentially opening up a new field for future human inner ear research.

Glomerulonephritis With Masked Monotypic Immunoglobulin Deposits and Concurrent Lymphomatous Infiltration.

Kidney injury can be a complication of hematopoietic neoplasia by both direct and indirect mechanisms. Virtually all lymphomas and plasma cell dyscrasias can show kidney involvement, including parenchymal infiltration and by secondary injury. Recently, a unique form of glomerulonephritis with masked monotypic immunoglobulin deposits has been reported, which shows frequent association with hematopoietic neoplasia and a propensity for progressive kidney disease. In many instances, these cases are likely diagnosed as glomerulonephritis with dominant C3 due to the absence of immunoglobulin staining by routine immunofluorescence microscopy. The patient reported here showed lymphomatous infiltration on kidney biopsy and mesangial proliferative glomerulonephritis with dominant staining for C3 without immunoglobulins on initial immunofluorescence; however, monotypic immunoglobulin G κ light chain was revealed after additional immunofluorescence staining was performed on the paraffin-embedded tissue. This patient's case highlights the evolving state of kidney biopsy interpretation and the expanding spectrum of kidney disease in the setting of hematopoietic neoplasia.

The effect of DNA damage on the pattern of immune-detectable DNA methylation in mouse embryonic fibroblasts.

The methylation of cytosine at CpG dinucleotides (5 meC) is an important epigenetic mechanism that governs genome stability and gene expression. Important ontological and pathological transitions are associated with marked global changes in detectable levels of methylation. We have previously found two pools of immune-detectable 5 meC exist within cells, a pool that can be detected after acid treatment of fixed cells to denature chromatin and another large but variable pool that requires a further tryptic digestion step for complete epitope retrieval. The trypsin-sensitive pool has been shown to be largely associated with the heterochromatic fraction (by a heterochromatin marker, HP1-ß) of the genome, and the size of this pool varies with the growth disposition of cells. Since DNA damage imposes large changes on chromatin structure the present study analyzed how such changes influences the faithful immunological detection of 5 meC within mouse embryonic fibroblasts. DNA damage was induced by either UV-irradiation or doxorubicin treatment, each of which resulted in increased levels of immune-detectable 5 meC at 24-48 h after treatment. There was a marked trypsin-sensitive pool of 5 meC in these cells which was significantly increased after DNA damage. The increased levels of 5 meC staining predominantly co-located with heterochromatic foci within nuclei, as assessed by HP1-ß staining. The relative amount of masked 5 meC after DNA damage was positively associated with increased levels of HP1-ß. The methyl binding protein, MBD1, was a less reliable measure of changes in 5 meC, with a significant fraction of 5 meC not being marked by MBD1. The cyto-epigenetic approaches used here reveal dynamism in the levels and localization of immune-detectable 5 meC within the nuclei of fibroblasts in response to DNA damage.

Introduction of Zinc-salt Fixation for Effective Detection of Immune Cell-related Markers by Immunohistochemistry.

Tissue localization of immune cells is critical to the study of disease processes in mouse models of human diseases. However, immunohistochemistry (IHC) for immune cell phenotyping in mouse tissue sections presents specific technical challenges. For example, CD4 and CD8 have been difficult to detect using IHC on formalin-fixed and paraffin-embedded mouse tissue, prompting alternative methods. We investigated the use of formalin-free zinc-salt fixation (ZN) and optimized IHC protocols for detecting a panel of immune cell-related markers (CD3, CD4, CD8, Foxp3, B220, F4/80, CD68, and major histocompatibility complex [MHC] class-I, MHC class-II, and Gr-1). The IHC results for these markers were compared on mouse spleen tissue treated with neutral buffered formalin (NBF) or ZN with or ZN without antigen retrieval (AR). Whereas CD4 and CD8 were not detected in NBF-treated tissue, all markers were detected in ZN-treated tissue without AR. Thus, the use of ZN treatment for IHC staining can be a good tool for studying immunoreactive lesions in tissues.

Antigen retrieval, blocking, detection and visualisation systems in immunohistochemistry: a review and practical evaluation of tyramide and rolling circle amplification systems.

To achieve specificity and sensitivity using immunohistochemistry it is necessary to combine the application of validated primary antibodies with optimised pre-treatment, detection and visualisation steps. The influence of these surrounding procedures is reviewed. A practical evaluation of tyramide signal amplification and rolling circle amplification detection methods is provided in which formalin fixed paraffin embedded sections of adenocarcinomas of breast, colon and lung together with squamous metaplasia of lung were immunostained with CD20 and CK19 primary antibodies. The results indicate that the detection systems are of comparable sensitivity and specificity.

When tissue antigens and antibodies get along: revisiting the technical aspects of immunohistochemistry--the red, brown, and blue technique.

Once focused mainly on the characterization of neoplasms, immunohistochemistry (IHC) today is used in the investigation of a broad range of disease processes with applications in diagnosis, prognostication, therapeutic decisions to tailor treatment to an individual patient, and investigations into the pathogenesis of disease. This review addresses the technical aspects of immunohistochemistry (and, to a lesser extent, immunocytochemistry) with attention to the antigen-antibody reaction, optimal fixation techniques, tissue processing considerations, antigen retrieval methods, detection systems, selection and use of an autostainer, standardization and validation of IHC tests, preparation of proper tissue and reagent controls, tissue microarrays and other high-throughput systems, quality assurance/quality control measures, interpretation of the IHC reaction, and reporting of results. It is now more important than ever, with these sophisticated applications, to standardize the entire IHC process from tissue collection through interpretation and reporting to minimize variability among laboratories and to facilitate quantification and interlaboratory comparison of IHC results.

Delineation of the healthy rabbit tongue by immunohistochemistry - A technical note.

In the oral cavity the tongue is an important muscular organ that supports the swallowing of food and liquids. It is responsible for the sense of taste, based on the many different taste buds it contains. Research in the field of tongue diseases demands for suitable preclinical models. The healthy rabbit tongue may therefore serve as baseline and reference for the pathological situation. With this consideration, we covered the fixation and histological stainings as well as the immunohistochemical labelling of the healthy rabbit tongue. In this technical note, initial choice of the fixative is discussed, with a comparison of formalin fixation and subsequent paraffin embedding versus cryopreservation. Moreover, we delineate the effect of an antigen retrieval step for formalin fixation by several examples. Finally, we provide ECM markers collagen I, collagen III, fibronectin, α-SMA and elastin staining as well as ki67 for proliferative status and PAR-2 protein expression as a marker for inflammatory status and nociception in tongue sections, mainly from the tongue body. Technically, we found superiority of paraffin sections for collagen I, collagen III, fibronectin, ki67 and α-SMA labelling, for selected detections systems. As for ECM components, the lamina propria was very rich in collagen and fibronectin, while the muscular body of the tongue showed only collagen and fibronectin positive areas between the muscle fibers. Moreover, α-SMA was clearly expressed in the walls of arteries and veins. The inflammatory marker PAR-2 on the other hand was prominently expressed in the salivary glands and to some extent in the walls of the vessels. Particular PAR-2 expression was found in the excretory ducts of the tongue. This technical note has the aim to provide baseline images that can be used to compare the pathological state of the diseased rabbit tongue as well as for inter-species comparison, such as mouse or rat tongue. Finally, it can be used for the comparison with the human situation.

Delineation of the healthy rabbit duodenum by immunohistochemistry - A short communication.

The duodenum acts as a vital organ that performs fundamental physiological functions like digestion and nutrient absorption. Situated in the lower abdomen, the duodenum is located between the stomach and the jejunum. Usually, the duodenum is divided into four anatomical portions. We here compare paraffin embedded and cryosections of the healthy rabbit duodenum for research purposes. This analysis evaluates the differential outcomes resulting from the application of these fixation methodologies in conjunction with immunohistochemical assays targeting extracellular matrix markers collagen I, collagen III, fibronectin, α-smooth muscle actin (α-SMA), and proliferation marker ki67 as well as inflammatory marker PAR-2. Subsequent recommendations are provided based on our findings. Furthermore, the advantage of an antigen retrieval step in immunohistochemical labelling of paraffin sections was demonstrated and confirmed with an isotype negative control. Basic classical histological stainings as HE, GT and elastin were also performed. Comparison of different stainings and labellings was performed in serial sections, showing that adjacent to the circular muscle of the duodenum, the connective tissue was composed of collagen I and fibronectin, while the artery and vein walls were predominantly α-SMA positive. Moreover, PAR-2 immunohistochemical staining was performed, where particularly a type of gland adjacent to Brunner's glands showed prominent PAR-2 positive areas, while the Brunner's glands themselves were PAR-2 negative. Proliferating ki67 positive cells facing the lumen were highly abundant in all kinds of glands except for the Brunner's glands. This effort serves to furnish the research community with reference imagery pertinent to scientists opting for the rabbit duodenum model. The diversity of staining techniques employed herein establishes a foundational repository of images, primed for comparative analysis against pathological conditions. Furthermore, these images hold the potential to illustrate inter-species variations. For instance, they can be juxtaposed against murine or rat intestinal tracts, or even offer insights into the human context.

Diagnostic Utility of Immunofluorescence on Formalin-Fixed Paraffin-Embedded Skin Biopsy.

Background: When immunofluorescence on the frozen section is insufficient or unavailable, salvage immunofluorescence techniques can be used on formalin-fixed, paraffin-embedded tissue. The goal of the current investigation was to evaluate the diagnostic value of paraffin immunofluorescence following proteinase K digestion on skin biopsy samples in comparison to fresh frozen immunofluorescence. Materials and Methods: It was standardized and compared to the immunofluorescence on fresh frozen tissue (IF-Frozen) for paraffin immunofluorescence by proteinase K digestion of formalin-fixed paraffin-embedded skin biopsies (IF-FFPE). The study included 50 native skin biopsy cases, and fluorescein isothiocyanate-labeled IgA, IgG, IgM, and C3 intensity levels were evaluated in each case. Results: A total of 50 cases of native skin biopsy were included in the study, and their intensities for IgA, IgG, IgM, and C3 antibodies were compared. The average staining intensities in each disease group for the antibodies had equal intensity or had a minor difference (1+)/significant difference (2+). Paraffin immunofluorescence, proteinase K digestion had the best correlation, that is, had either equal or minor difference (1+) with fresh frozen immunofluorescence. The difference of 2+ intensity of antibodies between IF-FFPE and IF-Frozen was noted mainly in C3 antibody on paraneoplastic pemphigus. IF-FFPE showed a sensitivity of 100%, 97.6%, 100%, and 81.6% for IgA, IgG, IgM, and C3, respectively, whereas the specificity was 100% for IgA, IgG, IgM, and C3. Limitations: Small sample size and and the employment of one method of antigen retrieval in IF-FFPE. Conclusion: Immunofluorescence techniques done on formalin-fixed paraffin-embedded tissue can serve as salvage techniques in cases where immunofluorescence on the frozen section may not be adequate or may not be available.