Epitope Retrieval Methods Research Articles

A comparative analysis of two antigen retrieval techniques: Microwave oven and pressure cooker for immunoexpression of estrogen and progesterone receptors in breast cancer tissue

Objectives: Antigen retrieval (AR) is an important step in Immunohistochemistry (IHC) which is used to unmask the antigenic sites and facilitate antigen-antibody binding. Adequate fixation of tissue is necessary to achieve consistent demonstration of tissue antigens that can be masked by the chemical process involved in formalin fixation and tissue processing. Out of the various methods of AR, heat-induced epitope retrieval (HIER) methods have greatly improved the quality and reproducibility of IHC. In this study, a comparison of the two most commonly used HIER methods-pressure cooker and microwave oven was done on thirty cases of breast carcinoma. Materials and Methods: Appropriate tumor sections were taken and subjected to manual IHC testing for estrogen receptor (ER) and progesterone receptor (PR) receptors in each case. The results were divided into technique and microscopy-based. The parameters assessed on microscopy were uniformity of nuclear staining, quality of nuclear staining, internal control staining, presence of background staining, and Allred score. The sensitivity and specificity and positive and negative predictive values for each method were calculated. Results: The parameters assessed on microscopy were comparable for both methods. Using a microwave oven, the sensitivity and specificity for ER and PR were 94% and 100%, respectively. Using a pressure cooker, the sensitivity, and specificity for ER were 94% and 100%, respectively, and for PR were 88% and 100%, respectively. On technical aspects, the pressure cooker method offers the advantage of being more convenient due to the possibility of simultaneous handling of more slides and being more time efficient. Conclusion: Both the AR methods had comparable results on microscopy. However, the pressure cooker has the benefit of being both time and money efficient from a technical standpoint.

Use of Citrus limon Juice as an Alternative Antigen Retrieval Solution for Formalin Fixed Breast Cancer Tissue in Immunohistochemistry

Background: The potential of Citrus limon fruit as a source of antigen retrieval agent is a significant step in the locally made brand of antigen retrieval solution with its availability and potential use in research and diagnosis. The cost of Immunohistochemistry tests is on the high side in Nigeria as a developing country, and many cancer patients have lost their lives in the cause of seeking alternative remedies to their prevailing health challenges due to financial constrain encountered in the cost of Immunohistochemistry (IHC) test. Its relevance makes the diagnosis of cancer effective for proper treatment and prognosis. Methods: Fifty (50) positive formalin-fixed breast tissues previously diagnosed using commercially prepared antigen retrieval solution (Thermoscientific – ephredia) were randomly selected for this study. Heat-induced epitope retrieval method (HIER) was used along with breast markers, namely Estrogen receptor (ER), Progesterone receptor (PR), and Human Epidermal Growth Factor Receptor 2 (HER-2) antibodies. Haematoxylin and eosin Techniave were used to demonstrate the general tissue structures to show the pleomorphic appearance of the cells invading the stroma, revealing an irregular shape with hyperchromatic nuclei. Results: Nuclear and cytoplasmic meshwork exposed without any background stain was observed. Estrogen receptor marker results in C. limon juice antigen retrieval was positive with a brown intensity score of 3 and proportion of stain score 5, having Thirty five (35) sections positive (70%). Progesterone receptor results in C. limon juice antigen retrieval revealed positive with a brown intensity score of 3 and a proportion of stain 5, with Forty-one (41) sections positive (82%). HER-2 results showed seventeen (17) sections (34%) positive with complete and intense brown circumferential membrane staining within >10% of tumor cells in C. limon juice antigen retrieval. Conclusion: The use of citrus juice is another source of antigen retrieval solution that can be explored for the immunohistochemistry test.

Frequency of overexpression of Her2/neu in colorectal adenocarcinomas– A hospital based cross-sectional study

Frequency of overexpression of Her2/neu in colorectal adenocarcinomas– A hospital based cross-sectional study - IJPO- Print ISSN No: - 2394-6784 Online ISSN No:- 2394-6792 Article DOI No:- 10.18231/j.ijpo.2020.006, Indian Journal of Pathology and Oncology-Indian J Pathol Oncol

Whole-mount Immunohistochemistry of Adult Zebrafish Retina for Advanced Imaging.

Immunohistochemistry is a widely used technique to examine the expression and subcellular localization of proteins. This technique relies on the specificity of antibodies and requires adequate penetration of antibodies into tissues. The latter is especially challenging for thick specimens, such as embryos and other whole-mount preparations. Here we describe an improved method of immunohistochemistry for retinal whole-mount preparations. We report that a cocktail of three reagents, Triton X-100, Tween-20, and DMSO, in blocking and antibody dilution buffers strongly enhances immunolabeling in whole-mount retinas from adult zebrafish. In addition, we establish that in whole retinal tissues, a classic epitope retrieval method, based on citrate buffer, is effective for immunolabeling membrane-associated proteins. Overall, this simple modification allows precise and reproducible immunolabeling of proteins in retinal whole-mounts.

Interrogating the Spatial Architecture of Human Bone Marrow Via Imaging Mass Cytometry

Hematopoietic stem cell (HSC) proliferation, self-renewal, differentiation, and trafficking are dependent, in part, upon signals generated by stromal cells in the bone marrow. Stromal cells are organized into niches that support specific subsets of hematopoietic progenitors. Intimate interactions between HSCs and neighboring stromal cells coordinate hematopoietic responses during periods of physiologic stress, while also maintaining the lifelong integrity of the hematopoietic stem cell pool. Hematopoietic niches are comprised of a heterogeneous population of stromal and hematopoietic cells. The identity and function of the known stromal cell subsets have primarily been gleaned through genetic manipulation of mouse models. In humans, the spatial organization of these stromal cells in bone marrow and the signals they generate to regulate hematopoiesis are poorly understood. Current methods to characterize bone marrow mesenchymal stromal cells in humans include: 1) immunostaining of bone sections; 2) analysis of flow sorted stromal cells; and 3) analysis of ex vivoexpanded mesenchymal stem/progenitors. Major limitations to all of these approaches exist that relate to the heterogeneity of bone marrow stromal cells, the lack of markers that reliably distinguish different stromal cell populations, and inherent technical limitations of the assays, such as the number of markers that can be analyzed at one time. Here we report our efforts to use imaging mass cytometry imaging (IMC) to interrogate the complex cellular architecture of human bone marrow. IMC allows for the simultaneous detection of up to 40 markers through the use of antibodies conjugated to elemental metal tags acquired by time-of-flight mass spectrometry. We first used standard immunostaining techniques to develop a panel of antibodies compatible with archived formalin-fixed paraffin-embedded (FFPE) human bone marrow specimens using a heat-induced epitope retrieval method. Hematopoietic lineages have been successfully identified using CD11b, CD68, CD15, CD14, CD16, CD11c, CD20, CD3, CD4, CD8a, CD38, CD45RA, CD45RO, CD235a, CD71, CD34, CD31; stromal cells and structures with CXCL12, alpha-smooth muscle actin, collagen I, vimentin; and nuclear staining using Ki67, Histone H3, and DNA intercalator. We used panels consisting of up to 18 of these antibodies each to test IMC on FFPE human bone marrow specimens. Imaging was performed using the Hyperion Imaging System (Fluidigm), which consists of a UV laser scanning module to capture regions of more than 10,000 cells with 1-micron resolution coupled with a Helios Mass Cytometer. Image analysis was performed by creation of a cell segmentation mask using CellProfiler software and high dimensional analysis in histoCAT. Representative images and corresponding dimensional reduction with t-SNE are shown in Figure 1A-H, demonstrating successful discrimination of distinct hematopoietic lineages. Gating on subpopulations within the t-SNE clusters can be projected on to the original image to illustrate spatial distribution and marker co-expression, as an example CD3+ and CD8+ cells are shown in Figure 1I. These data indicate that IMC allows for highly multiplexed analysis of bone marrow cell populations. Developing imaging techniques for analysis of tissue-banked FFPE bone marrow samples would have broad applications for translational research on hematologic diseases. In particular, this technology has tremendous potential to advance understanding of the spatial architecture of human bone marrow and to investigate alterations in the bone marrow environment in malignant hematopoiesis. Disclosures Oh: Incyte: Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy.

Antibody Selection for Cancer Target Validation of FSH-Receptor in Immunohistochemical Settings.

Background: The follicle-stimulating hormone (FSH)-receptor (FSHR) has been reported to be an attractive target for antibody therapy in human cancer. However, divergent immunohistochemical (IHC) findings have been reported for FSHR expression in tumor tissues, which could be due to the specificity of the antibodies used. Methods: Three frequently used antibodies (sc-7798, sc-13935, and FSHR323) were validated for their suitability in an immunohistochemical study for FSHR expression in different tissues. As quality control, two potential therapeutic anti-hFSHR Ylanthia® antibodies (Y010913, Y010916) were used. The specificity criteria for selection of antibodies were binding to native hFSHR of different sources, and no binding to non-related proteins. The ability of antibodies to stain the paraffin-embedded Flp-In Chinese hamster ovary (CHO)/FSHR cells was tested after application of different epitope retrieval methods. Results: From the five tested anti-hFSHR antibodies, only Y010913, Y010916, and FSHR323 showed specific binding to native, cell-presented hFSHR. Since Ylanthia® antibodies were selected to specifically recognize native FSHR, as required for a potential therapeutic antibody candidate, FSHR323 was the only antibody to detect the receptor in IHC/histochemical settings on transfected cells, and at markedly lower, physiological concentrations (ex., in Sertoli cells of human testes). The pattern of FSH323 staining noticed for ovarian, prostatic, and renal adenocarcinomas indicated that FSHR was expressed mainly in the peripheral tumor blood vessels. Conclusion: Of all published IHC antibodies tested, only antibody FSHR323 proved suitable for target validation of hFSHR in an IHC setting for cancer. Our studies could not confirm the previously reported FSHR overexpression in ovarian and prostate cancer cells. Instead, specific overexpression in peripheral tumor blood vessels could be confirmed after thorough validation of the antibodies used.

A Method for the Immunohistochemical Identification and Localization of Osterix in Periosteum-Wrapped Constructs for Tissue Engineering of Bone.

A novel immunohistochemistry (IHC) approach has been developed to label and localize osterix, a bone-specific transcription factor, within formalin-fixed, paraffin-embedded, tissue-engineered constructs uniquely containing synthetic polymers and human periosteal tissue. Generally, such specimens consisting in part of polymeric materials and mineral are particularly difficult for IHC identification of proteins. Samples here were fabricated from human periosteum, electrospun poly-l-lactic acid (PLLA) nanofibers, and polycaprolactone/poly-l-lactic acid (PCL/PLLA, 75/25) scaffolds and harvested following 10 weeks of implantation in athymic mice. Heat-induced and protease-induced epitope retrieval methods from selected existing protocols were examined to identify osterix. All such protease-induced techniques were unsuccessful. Heat-induced retrieval gave positive results for osterix immunohistochemical staining in sodium citrate/EDTA/Tween 20 with high heat (120C) and pressure (~30 psi) for 10 min, but the heat and pressure levels resulted in tissue damage and section delamination from slides that limited protocol effectiveness. Heat-induced epitope retrieval led to other osterix-positive staining results achieved with minimal impact on structural integrity of the tissue and polymers in sodium citrate/EDTA/Tween 20 buffer at 60C and normal pressure (14.5 psi) for 72 hr. The latter approach identified osterix-positive cells by IHC within periosteal tissue, layers of electrospun PLLA nanofibers, and underlying PCL/PLLA scaffolds of the tissue-engineered constructs.

Diagnostic Immunohistochemistry: What Can Go Wrong and How to Prevent It.

-There are a number of critical factors that can lead to incorrect results if the diagnostic pathologist performing immunohistochemistry is unaware of, or not vigilant about, their influence. -To highlight 3 arenas in which errors may be introduced. -For choosing the correct primary antibody, selection of the most appropriate antibodies for a given clinical application can be aided by obtaining information from the vendor; however, this can yield incomplete information. There are a number of online databases that have comparisons of antibodies from different vendors, particularly with respect to their use and properties. Reading the published literature can assist in this process, particularly with respect to determining antibody sensitivity and specificity, but it is a daunting task to keep up with all of the immunohistochemistry-related papers published. Finally, Web sites of a number of quality assurance organizations are accessible and can provide a wealth of information comparing the "real world" performance characteristics of different antibodies to the same target protein. False-positive signals can result from a number of factors, including the use of inappropriately high antibody concentration, and "pseudospecific" signal that is in the wrong compartment of the cell. False-negative signal can result from factors such as use of a nonoptimized epitope retrieval method. It is critical that epitope retrieval methods be optimized for each antibody employed in the laboratory. -By paying attention to these potential problems, the "black box" of diagnostic immunohistochemistry can be made more transparent.

A simple and effective heat induced antigen retrieval method

In this paper, we describe an additional step to the standard method of heat induced antigen retrieval to improve the detection of antibody staining of formalin fixed paraffin embedded tissue sections. Direct heating of tissues in buffer is an efficient epitope retrieval method but often results in the damage or loss of tissues. In this modified method, before keeping in buffer for heating, we overlapped the tissue on the slide with a plain slide by clipping one end using a normal paperclip, keeping a minimum gap between the slides. Tissues heated in this way in buffer had following advantages over normal heat treatment for epitope retrieval.•Tissues were intact even at high temperatures which improved the quality of staining by preventing fold, damage or detachment of tissues from the slides.•The method is very safe and economical compared to the methods using microwave or pressure cooker.•This simple method also appears to be very effective and less time consuming compared to the existing methods.

Study in ER/PR and HER2 receptor IHC

Recently, the development of Estrogen Receptor (ER), Progesterone Receptor (PR), and Human Epidermal Growth Factor Receptor 2 (HER2) receptors in immunohistochemistry has amassed significant prognostic value in the treatment of breast carcinomas. Its introduction provides an inexpensive and widely available testing protocol yielding vital pathological information regarding patient outcome and therapeutic response. However, in conjunction with its continued advancement, these markers withstand increased hindrance in reaching their full diagnostic potential due to the lack of a standardized protocol. The primary focus of this study was to develop standardized protocols for each breast marker. Through manipulation of antigen retrieval methods and dilutions, highly accurate and reproducible staining results were achieved. Validations of ER and PR were run with alternating Heat Induced Epitope Retrieval (HIER) methods using solutions buffered with EDTA and citrate. Simultaneous comparisons were run using the laboratory's Ventana platform, establishing parallels between previously validations and new optimizations to ease antibody transition. HER2 optimization initial workups included Dako, Thermo Fisher, and Ventana antibodies monitored over extended time periods . The finalized protocol utilizes Ventana's HER2 antibody validated to run on a Leica platform. The addition of an alternative hematoxylin counterstain and citric acid clarifier was applied in order to enhance staining clarity and ease of quantitation. The use of Ventana diluents in working antibody solution, restrictions on quantity made, and refrigeration, may create staining consistency. The laboratory used was able to offer a diagnostically significant receptor panel with minimal variations between cases, having an enormously positive impact on patient care.

An Update on Immunohistochemistry in Translational Cancer Research

Immunohistochemistry (IHC) takes advantage of the specific binding between antigen and antibody to measure the presence and abundance of antigen while simultaneously providing morphologic context on a tissue section. Since the revolutionary application of heat-induced epitope retrieval methods on formalin-fixed paraffin-embedded tissues, which started in early 1990s, IHC has been routinely used in diagnostic pathology. This approach has also enabled mining of the rich archives of pathologic specimens for exploration in translational cancer research. Newer IHC biomarkers are being continuously found as aids in differential diagnosis, prediction of outcome or response to molecular-targeted therapies. These are prime examples for translational cancer research. The last decade has witnessed some significant improvements in the use of this technology. This review provides an overview on the current status of IHC as applied in translational cancer research, commenting on the underlying principles in specimen preparation, reagent choice, staining procedure, and results evaluation so that both beginners and seasoned users could appreciate the key factors and benefit from this update.

The prevalence of immunohistochemically determined oestrogen receptor positivity in primary breast cancer is dependent on the choice of antibody and method of heat-induced epitope retrieval – prognostic implications?

Background. Oestrogen receptor (ER) status is important for the choice of systemic treatment of breast cancer patients. However, most data from randomised trials on the effect of adjuvant endocrine therapy according to ER status are based on the cytosol methods. Comparisons with immunohistochemical methods have given similar results. The aim of the present study was to examine whether different ER antibodies and heat-induced epitope retrieval (HIER) methods influence the prevalence of ER-positivity in primary breast cancer. Material and methods. This study is based on patients included in a clinical trial designed to compare the effect of two years of adjuvant tamoxifen versus no adjuvant systemic treatment in premenopausal women. From 1986 to 1991, 564 patients from two study centres in Sweden were enrolled and randomised. Patients were randomised independently of ER status. In the present study, ER status was assessed on tissue microarrays with the three different ER antibody/HIER combinations: 1D5 in citrate pH 6 (n = 390), SP1 in Tris pH 9 (n = 390) and PharmDx in citrate pH 6 (n = 361). Results. At cut-offs of 1% and 10%, respectively, the prevalence of ER-positivity was higher with SP1 (75% and 72%) compared with 1D5 (68% and 66%) and PharmDx (66% and 62%). At these cut-offs, patients in the discordant groups (SP1-positive and 1D5-negative) seem to have a prognosis intermediate between those of the double-positive and double-negative groups. Comparison with the ER status determined by the cytosol-based methods in the discordant group also showed an intermediate pattern. The repeatability was good for all antibodies and cut-offs, with overall agreement ≥ 93%. Conclusion. The present study shows that the choice of antibody and HIER method influences the prevalence of ER-positivity. We suggest that this be taken into consideration when choosing a cut-off for clinical decision making.

Evidence of interstitial Cajal-like cells in human gallbladder

The aim of this study was to assess the presence of interstitial Cajal-like cells (ICLCs) in the human gallbladder and to determine their distinctive characteristics on the basis of double immunohistochemical staining. Gallbladder specimens were obtained from 30 patients subjected to elective laparoscopic cholecystectomy for symptomatic gallstone disease. Tissue samples were fixed in 4% phosphate-buffered paraformaldehyde, processed, embedded in paraffin, and, after sectioning, routinely stained with HE. Tissue antigens were retrieved using the heat-induced epitope retrieval (HIER) method. For simultaneous visualisation of two antigens, an indirect double immunofluorescence procedure was applied. ICLCs were defined as CD117-immunopositive and tryptase-immunonegative objects. They were predominantly fusiform in shape with sparse branches that were visible in some sections. ICLCs were observed throughout the organ including the gallbladder's fundus, body (corpus) and neck, being most numerous in the corpus. The ICLCs were detected almost exclusively within the muscularis propria and they were arranged parallel to smooth muscle cells. The following subpopulations of ICLCs were observed: ICLC-IM (intramuscular ICLCs) localised between smooth muscle fibres forming one muscle bundle; and ICLC-IB (interbundle ICLCs) localised within the connective tissue separating smooth muscle bundles. Thus, the presence of ICLCs in the human gallbladder was clearly identified, demonstrated by double immunohistochemistry which was found to be a reliable method for differentiating ICLCs from mast cells.

Evidence of interstitial Cajal-like cells in human gallbladder

The aim of this study was to assess the presence of interstitial Cajal-like cells (ICLCs) in the human gallbladder and to determine their distinctive immunophenotypic characteristics on the basis of double immunohistochemical staining. Gallbladder specimens investigated in the study were derived from patients (8 males, mean age 51.9±10.7 y; 22 females, mean age 52.9±15.1 y) subjected to elective laparoscopic cholecystectomy for symptomatic gallstone disease. Tissue samples were fixed in 4% phosphate-buffered paraformaldehyde, processed and embedded in paraffin. Sections from gallbladders were routinely stained with hematoxylin and eosin. Tissue antigens were retrieved using heat-induced epitope retrieval (HIER) method. For simultaneous visualization of two antigens an indirect double immunofluorescence procedure was applied. Primary antibodies used in this study were rabbit anti-c-Kit (anti-CD117) and mouse anti-mast cell tryptase. ICLCs were defined as CD117-immunopositive and tryptase-immunonegative objects. They were predominantly fusiform in shape with sparse branches that were visible in some sections. ICLCs were observed throughout the gallbladder, including the fundus, body (corpus) and neck, although they were most numerous in the corpus. The ICLCs were detected almost exclusively within the muscularis propria and they were arranged parallel to smooth muscle cells. The following subpopulations of the ICLCs were observed: ICLC-IM (intramuscular ICLCs) localized between smooth muscle fibers forming one muscle bundle and ICLC-IB (interbundle ICLCs) localized within the connective tissue separating smooth muscle bundles. In the current study we confirmed the presence of ICLCs in the human gallbladder. Applied double immunohistochemistry was found to be a reliable method for identifying ICLCs and for differentiating them from mast cells.

Using Unfixed, Frozen Tissues to Study Natural Mucin Distribution

Mucins are complex and heavily glycosylated O-linked glycoproteins, which contain more than 70% carbohydrate by weight1-3. Secreted mucins, produced by goblet cells and the gastric mucosa, provide the scaffold for a micrometers-thick mucus layer that lines the epithelia of the gut and respiratory tract3,4. In addition to mucins, mucus layers also contain antimicrobial peptides, cytokines, and immunoglobulins5-9. The mucus layer is an important part of host innate immunity, and forms the first line of defense against invading microorganisms8,10-12. As such, the mucus is subject to numerous interactions with microbes, both pathogens and symbionts, and secreted mucins form an important interface for these interactions. The study of such biological interactions usually involves histological methods for tissue collection and staining. The two most commonly used histological methods for tissue collection and preservation in the clinic and in research laboratories are: formalin fixation followed by paraffin embedding, and tissue freezing, followed by embedding in cryo-protectant media.Paraffin-embedded tissue samples produce sections with optimal qualities for histological visualization including clarity and well-defined morphology. However, during the paraffin embedding process a number of epitopes become altered and in order to study these epitopes, tissue sections have to be further processed with one of many epitope retrieval methods13. Secreted mucins and lipids are extracted from the tissue during the paraffin-embedding clearing step, which requires prolong incubation with organic solvents (xylene or Citrisolv). Therefore this approach is sub-optimal for studies focusing on the nature and distribution of mucins and mucus in vivo.In contrast, freezing tissues in Optimal Cutting Temperature (OCT) embedding medium avoids dehydration and clearing of the sample, and maintains the sample hydration. This allows for better preservation of the hydrated mucus layer, and thus permits the study of the numerous roles of mucins in epithelial biology. As this method requires minimal processing of the tissue, the tissue is preserved in a more natural state. Therefore frozen tissues sections do not require any additional processing prior to staining and can be readily analyzed using immunohistochemistry methods.We demonstrate the preservation of micrometers-thick secreted mucus layer in frozen colon samples. This layer is drastically reduced when the same tissues are embedded in paraffin. We also demonstrate immunofluorescence staining of glycan epitopes presented on mucins using plant lectins. The advantage of this approach is that it does not require the use of special fixatives and allows utilizing frozen tissues that may already be preserved in the laboratory.

KAI1/CD82 is a novel target of estrogen receptor-mediated gene repression and downregulated in primary human breast cancer (Int J Cancer 123: 2239-2246, 2008)

With interest I read the article by Christgen et al. that was recently published under the Fast Track heading in the International Journal of Cancer.1 The major reported findings, nicely summarized in the article's title, are that (a) KAI1/CD82 is downregulated in 77% of human breast carcinomas and (b) KAI1 downregulation is strongly correlated with ER positivity. I do not question the accuracy of the reported data. However, I take exception to the authors' claim, repeated several times throughout their article, that their major findings are novel. The fact is that my colleagues and I published practically identical observations 3 years ago.2 Specifically, we reported downregulation of KAI1 expression in 74 of 98 (76%) archival breast carcinomas, and we mentioned that KAI1 downregulation was significantly more common in ER-positive (48 of 55, 87%) versus ER-negative (22 of 36, 61%) tumors (p = 0.0052). Although the focus of our manuscript was on new cell membrane biomarkers involved in breast oncogenesis, we very clearly detailed our observations on KAI1 expression in human breast cancer tissues and 9 cell lines including MCF-7, which Christgen used as a negative control. We published our data in Clinical Cancer Research, which can hardly be considered an obscure journal. It is hard to imagine that even a cursory review of the literature involving KAI1/CD82 and breast cancer would not have brought our article to Christgen et al.'s attention. A scientific finding does not become novel by ignoring its previous description by other investigators. It would have been appropriate for Christgen etal. to acknowledge our published data. Of course, in so doing they would have had to admit that most of their data are confirmatory rather than novel, notwithstanding the fact that they documented the inverse relationship between ER and KAI1 expression in greater detail. Christgen et al. also give the impression that their immunohistochemical staining method for KAI1 is a major technological advance. In fact, they used the same anti-KAI1 antibody and the same heat induced epitope retrieval method that we published in 2005.2 Already in 1999, we had demonstrated that KAI1 protein expression can reliably be evaluated in fixed tissues by immunohistochemistry using a different antibody.3 Christgen also confirms our original observation that KAI1 expression is not correlated with axillary lymph node involvement, tumor grade, or c-erbB-2 status.2 Christgen et al. describe several in vitro experiments as well. They show that the growth and migration of ER-positive, but not ER-negative, breast cancer cells is inhibited by fulvestrant, an antiestrogenic agent, and that this is correlated with up- or downregulation of 4 indicator genes. The authors acknowledge that those experiments confirm previously published observations by other research groups.4-7 It seems that Christgen et al.'s only original and novel contribution is the finding that KAI1 is upregulated by fulvestrant in 2 ER-positive breast cancer cell lines. The International Journal of Cancer may consider following the example of other scientific journals that discourage authors from inserting superlatives (“first”, “largest”, etc.) into their manuscripts. Some of those claims do not stand up to scrutiny. Evidently the reviewers assigned to Christgen's article were not sufficiently familiar with the literature to rebut the authors' exaggerated claims of novelty. In conclusion, I believe the data presented by Christgen et al. are interesting and a useful addition to the scientific literature. However, their major findings are confirmatory (rather than novel) in nature. It is inappropriate for the authors to suggest that their methodology and experimental results are groundbreaking, when they essentially replicate portions of an article that they unfortunately chose not to cite. Yours sincerely, Joseph Geradts MD

Evaluation of α-synuclein immunohistochemical methods used by invited experts

The use of alpha-synuclein immunohistochemistry has altered our concepts of the cellular pathology, anatomical distribution and prevalence of Lewy body disorders. However, the diversity of methodology between laboratories has led to some inconsistencies in the literature. Adoption of uniformly sensitive methods may resolve some of these differences. Eight different immunohistochemical methods for demonstrating alpha-synuclein pathology, developed in eight separate expert laboratories, were evaluated for their sensitivity for neuronal elements affected by human Lewy body disorders. Identical test sets of formalin-fixed, paraffin-embedded sections from subjects diagnosed neuropathologically with or without Lewy body disorders were stained with the eight methods and graded by three observers for specific and nonspecific staining. The methods did not differ significantly in terms of Lewy body counts, but varied considerably in their ability to reveal neuropil elements such as fibers and dots. One method was clearly superior for revealing these neuropil elements and the critical factor contributing to its high sensitivity was considered to be its use of proteinase K as an epitope retrieval method. Some methods, however, achieved relatively high sensitivities with optimized formic acid protocols combined with a hydrolytic step. One method was developed that allows high sensitivity with commercially available reagents.

Immunohistochemical markers for quantitative studies of neurons and glia in human neocortex.

Reproducible visualization of neurons and glia in human brain is essential for quantitative studies of the cellular changes in neurological disease. However, immunohistochemistry in human brain specimens is often compromised because of prolonged fixation. To select cell lineage-specific antibodies for quantitative studies of neurons and the major types of glia, we used 29 different antibodies, different epitope retrieval methods, and different detection systems to stain tissue arrays of formalin-fixed human brain. The screening pointed at CD45/leukocyte common antigen (LCA), CD68(KP1), 2',3' cyclic nucleotide phosphatase (CNPase), glial fibrillary acidic protein (GFAP), HLA-DR, Ki67, neuronal nuclei (NeuN), p25alpha-antigen, and S100beta as candidates for future cell counting purposes, because these markers visualized specific neuronal and glial cell bodies. However, significant negative correlation between staining result and formalin fixation was observed by blinded scoring of staining for CD45/LCA, CNPase, GFAP, and NeuN in brain specimens fixed by immersion and stored up to 10 years in 4% formalin solution at room temperature, independent of donor sex and postmortem interval. In contrast, improved preservation of NeuN and CNPase staining, and full preservation of GFAP and CD45/LCA staining in tissue fixed by perfusion and stored for up to 3 years in 0.1% paraformaldehyde solution at 4C, indicated that immunohistochemistry can be performed in well-preserved biobank material.

A focus on fixation

Three fixation issues related to immunostaining are discussed here: 1) Generally, a tissue block is fixed, then embedded and sectioned (pre-fixation). The type of fixative applied, crosslinking or coagulating, has an impact on selecting an epitope retrieval method. Individual antigens have a fixation–retrieval characteristic. 2) A long fixation time, especially with crosslinking fixatives, may compromise the result of immunostaining. This negative effect varies among different antigens and can be partially restored by applying a more sensitive/efficient detection system such as tyramide amplification. 3) Sections cut from a fresh frozen tissue block usually are acetone fixed (post-fixation). This was accepted as the “gold standard” for a long time. Post-fixation, however, may have serious consequences for preservation of small peptides leaking from the cut open cells, whereas this is not the case with pre-fixed intact cells. Consequently, the concept of an acetone post-fixed cryostat tissue section as “gold standard” no longer exists and a more appropriate use of the terms immunohistochemistry and immunocytochemistry therefore seems justified. For many antibodies, it is not known whether a formalin fixed, paraffin embedded tissue specimen is appropriate. Suggestions are made for creating a positive control cell block for testing such antibodies.

The brain-specific protein TPPP/p25 in pathological protein deposits of neurodegenerative diseases

Immunohistochemical detection of protein components of pathological inclusions is widely used for neuropathological diagnosis of neurodegenerative disorders. However, different antibodies and antigen unmasking methods may account for variability between research studies and thus may affect diagnostic accuracy. Using two different antibodies raised against either a segment (184-200 aa) or the full length of human recombinant brain-specific tubulin polymerization promoting protein TPPP/p25, we immunohistochemically screened neurodegenerative disorders, both with and without pathological alpha-synuclein structures. We tested three different epitope unmasking methods, we applied laser confocal microscopy to evaluate double immunolabelling, and we compared the amount of structures exhibiting TPPP/p25 and alpha-synuclein immunoreactivity. We demonstrate that there are a variety of staining patterns depending on the epitope retrieval method and antibody used. The antibody raised against aa 184-200 segment of TPPP/p25 is better in immunolabelling the majority of alpha-synuclein immunopositive neuronal and glial pathological profiles detectable in Parkinson's disease, diffuse Lewy-body disease, and multiple system atrophy, in addition to immunostaining some extracellular huntingtin immunoreactive structures, lipofuscin, and neuromelanin particles. In contrast, the one raised against the full-length human recombinant TPPP/p25 is more suitable to immunodetect normal oligodendrocytes. Exposition of the segment aa 184-200 of TPPP/p25 in the aggregates of pathological inclusions renders this antibody a reliable marker of all types of alpha-synucleinopathies and suggests a role for TPPP/p25 in the aggregation process of some neurodegenerative conditions.