Antigen Retrieval Method Research Articles

Equivalency of RT-PCR and immunohistochemistry: fact or factoid

Management of breast cancer relies heavily on determination of predictive and prognostic markers. Analysis of estrogen and progesterone receptors was originally performed using ligand-binding assays (LBA); this has been replaced by immunohistochemistry (IHC) in the last couple of decades. In recent days, RT-PCR assays for ER and PR have been proposed as a superior alternative to IHC. Several studies have attempted to analyze the concordance between these different types of assays. In this issue, investigators from the Hellenic Oncology Group examines the expression and predictive significance of ER, PR and microtubule-associated protein tau (MAP-tau) by quantitative RT-PCR (qRT-PCR) in a series of 274/279 breast cancer patients enrolled in clinical trial HE 10/97 [1]. In these analyses, MAP-tau strongly correlated with ER and PR mRNA status (Spearmann r = 0.52 and 0.64, P \ 0.001). However, they find only a modest correlation between RT-PCR and IHC results for ER (kappa = 0.41) and a fair correlation for PR (kappa = 0.33). Significantly, no correlation was observed between increasing ER and PR mRNA and increasing benefit from endocrine therapy in 203 ER and/or PR IHC-positive patients receiving adjuvant hormone therapy (Wald P = 0.54 for ER, 0.51 for PR). As has been acknowledged by the authors, the findings in this study are at variance with prior reports [2–4] with regards to concordance between IHC and qRT-PCR. These differences seen in the qRT-PCR results may be ascribed to a variety of pre-analytical and analytical parameters, which are briefly discussed below. Analysis for both these assays was performed on formalin fixed paraffin-embedded (FFPE) tissues obtained from multiple institutions; it can be safely presumed that these were associated with a variety of fixation and processing protocols. The time duration of warm ischemia, length of exposure to formalin, and exact temperatures used in tissue processing and embedding are thought to be important for intactness of both RNA and protein. In addition, the age of the paraffin block has been shown to directly correlate with amount of fragmentation of RNA [5]. Akin to the choice of antigen retrieval methods available for IHC, multiple kits are available for extraction of RNA from FFPE materials. Abramowitz et al. [6] have recently performed a comparative analysis of different commercially available kits and demonstrated significant differences in quantities of RNA obtained and reproducibility of results. In their study, variation in the digestion/ incubation time (3 h versus overnight) resulted in significant differences in reproducibility even when using the same kit. Tumor tissue is a composite mixture of tumor cells, tumor microenvironment and non-malignant elements, which include normal breast epithelium and in situ carcinoma. As morphologic assessment is a critical part of IHC methods, expression is only analyzed in tumor cells. The pathologist has the capacity to exclude expression in normal elements and ductal carcinoma in situ, which therefore do not contribute to the final results. Assays such as LBA and qRT-PCR that require tissue digestion, do not permit morphologic distinction of tumor cells from nonmalignant This is a commentary to doi:10.1007/s10549-008-0144-9.

Modeling formalin fixation and histological processing with ribonuclease A: effects of ethanol dehydration on reversal of formaldehyde cross-links

Understanding the chemistry of protein modification by formaldehyde fixation and subsequent tissue processing is central to developing improved methods for antigen retrieval in immunohistochemistry and for recovering proteins from formalin-fixed, paraffin-embedded (FFPE) tissues for proteomic analysis. Our initial studies of single proteins, such as bovine pancreatic ribonuclease A (RNase A), in 10% buffered formalin solution revealed that upon removal of excess formaldehyde, monomeric RNase A exhibiting normal immunoreactivity could be recovered by heating at 60°C for 30 min at pH 4. We next studied tissue surrogates, which are gelatin-like plugs of fixed proteins that have sufficient physical integrity to be processed using normal tissue histology. Following histological processing, proteins could be extracted from the tissue surrogates by combining heat, detergent, and a protein denaturant. However, gel electrophoresis revealed that the surrogate extracts contained a mixture of monomeric and multimeric proteins. This suggested that during the subsequent steps of tissue processing protein–formaldehyde adducts undergo further modifications that are not observed in aqueous proteins. As a first step toward understanding these additional modifications we have performed a comparative evaluation of RNase A following fixation in buffered formaldehyde alone and after subsequent dehydration in 100% ethanol by combining gel electrophoresis, chemical modification, and circular dichroism spectroscopic studies. Our results reveal that ethanol-induced rearrangement of the conformation of fixed RNase A leads to protein aggregation through the formation of large geometrically compatible hydrophobic β-sheets that are likely stabilized by formaldehyde cross-links, hydrogen bonds, and van der Waals interactions. It requires substantial energy to reverse the formaldehyde cross-links within these sheets and regenerate protein monomers free of formaldehyde modifications. Accordingly, the ethanol-dehydration step in tissue histology may be important in confounding the successful recovery of proteins from FFPE tissues for immunohistochemical and proteomic analysis.

Antigen retrieval with protease digestion applied in immunohistochemical diagnosis of Alport syndrome

Immunofluorescence (IF) staining of type IV collagen alpha chains on fresh frozen renal tissue is a convenient and accurate diagnosis technique for Alport syndrome (AS), which is restricted in the application with a non-frozen section. An alternative method for a paraffin-embedded section is needed in order to extend the application in various specimens. In this study, immunohistochemical staining of type IV collagen alpha chains on paraffin-embedded renal sections was investigated. Three antigen retrieval methods including autoclave heating, pepsin digestion and protease digestion were tried on paraffin-embedded renal sections from two X-linked male AS patients, two X-linked female AS patients and two autosomal recessive AS patients. Two patients with isolated haematuria were also involved. Normal portions of nephrectomized kidneys from two patients with renal tumours were used as controls. The immunohistochemical staining pattern of type IV collagen was compared with that of IF staining. The results showed that both the autoclave heating method and protease digestion can be used for antigen retrieval for type IV collagen alpha chains. Compared to autoclave heating, protease digestion had the advantage of being less time consuming, with less renal sections being lost from the slides; it was the most optimal antigen retrieval method in this study. After protease digestion for 40 min, type IV collagen alpha3 and alpha5 chains showed a clear and proper distribution pattern in AS patients, haematuria patients and controls, which is the same as that of IF staining. Compared to the autoclave heating method, protease antigen retrieval is more convenient and effective and can be used to restore type IV collagen alpha chains on paraffin-embedded renal sections for the diagnosis of AS.

Correlation between IHC and FISH for HER2/neu assessment in patients with breast cancer

20718 Background: HER-2/neu status of the breast cancer (BC) is determined by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). Numerous reports have been published identifying discrepancies between IHC and FISH methods from different manufacturers, as well as tissue processing, reagent variability, antigen retrieval methods, scoring interpretation, tumor heterogeneity, and the semiquantitative nature of the test. The objective was to compare the results of HER-2 assessment by IHC and FISH in patients (pts) with BC being considered for trastuzumab therapy. Methods: For HER2 status review, IHC was used with a policlonal antibody (CB11–1/500), antigenic recovery, Diaminobenzidina (DAKO) and system of detection ENVISION (DAKO). Manual method per protocol of work was used (National Plan Her2). The results were interpreted using score 0 to 3+. FISH was used to assess HER-2 gene amplification. The tumors were tested in two reference centres. All procedures were applied to de-paraffinised tissue sections of BC samples. Results: From July to December 2007, 42 cases of infiltrative BC were studied and correlation with HER-2 overexpression between IHC and FISH was determined. Three plus HER-2 overexpression was found in 9 pts (23%), 2+ in 8 (20%), 1+ in 3 (7,7%) and 0 in 19 (48,7%). In 3 cases this determination was not done. FISH was positive in 14 cases (33,3%) and negative in 28 (66,6%). Only 2/19 (10,5%) IHC score 0 were FISH positive. Four of 8 (50%) IHC 2+ were FISH positive and the other 4 were negative. Only 1 of the 9 IHC score 3+ was FISH negative, so the discordance in this case was 11,1%. Conclusions: We found a significant discordance in 3 cases. Two were IHC score 0 and FISH positive, and 1 was IHC score 3+ and FISH negative. In the 8 cases of IHC score 2+, FISH was used to define positivity: 4/8 (50%) were FISH positive. We suggest to carry out FISH to evaluate the status of HER-2 in BC, especially when is considering the use of trastuzumab therapy. No significant financial relationships to disclose.

Evaluation of microvascular density in tumors: pro and contra.

Microvascular density (MVD) counting protocols have become the morphological gold standard to assess the neovasculature in human tumors. This method requires the use of specific markers to vascular endothelium and of immunohistochemical procedures to visualize microvessels. MVD determined in primary tumors is significantly associated with metastasis and prognosis in several tumors and is most predictive in those tumors that induce significant angiogenesis, namely carcinomas of breast and prostate, and haematological malignancies. There is such a wide range of antibodies and suppliers, antigen retrieval methods, designation of high and low vessel count groups, patient study groups and data interpretation, that it is exceedingly difficult to compare results.

RE‐EVALUATION OF ARCHIVAL “DLDH” CASES FOR FRONTOTEMPORAL LOBAR DEGENERATION WITH UBIQUITIN INCLUSIONS: ANTIGEN RETRIEVAL IS ESSENTIAL

The neuropathologic subtypes of frontotemporal lobar degeneration (FTLD) can frequently be differentiated immunohistochemically. The identification of abnormal deposits of tau, ubiquitin, and, more recently, TAR DNA‐binding protein 43 (TDP‐43) and alpha‐internexin may allow separation of neuropathologic subtypes, whereas the lack of staining for these proteins may lead to a diagnosis of “dementia lacking distinctive histology” (DLDH). Because of the importance of accurate pathological diagnosis for the understanding of these disorders, we investigated whether a wider panel of antibodies and more sensitive immunohistochemical methods could allow for better neuropathologic characterization. We undertook a study of 13 autopsy cases with non‐specific diagnoses such as DLDH. We relabeled these brains with antibodies to tau, ubiquitin, TDP‐43, and alpha‐internexin. In addition, we tested the effect of antigen retrieval on the ability to detect the ubiquitin immunolabel. In 7 of the 13 cases, ubiquitin‐immunoreactive inclusions were identified. These cases also immunolabeled with the antibody to TDP‐43. This pattern of staining supported a reclassification to FTLD with ubiquitin inclusions (FTLD‐U). In all of these cases, the antigen retrieval method markedly enhanced ubiquitin immunolabel and was critical for the identification of FTLD‐U pathology. Supported by 1P50‐AG025688.

Assessment of α-Synuclein Pathology: A Study of the BrainNet Europe Consortium

To determine the reliability of assessment of alpha-synuclein-immunoreactive (alphaS-IR) structures by neuropathologists, 28 evaluators from 17 centers of BrainNet Europe examined current methods and reproducibility of alphaS-IR evaluation using a tissue microarray (TMA) technique. Tissue microarray blocks were constructed of samples from the participating centers that contained alphaS-IR structures. Slides from these blocks were stained in each center and assessed for neuronal perikaryal inclusions, neurites, and glial cytoplasmic inclusions. The study was performed in 2 phases. First, the TMA slides were stained with the antibody of the center's choice. In this phase, 59% of the sections were of good or acceptable quality, and 4 of 9 antibodies used performed consistently. Differences in interpretation and categorization of alphaS-IR structures, however, led to differing results between the laboratories. Prior to the second phase, the neuropathologists participated in a training session on the evaluation of alphaS-IR structures. Based on the results of the first phase, selected antibodies using designated antigen retrieval methods were then applied to TMA slides in the second phase. When the designated methods of both staining and evaluation were applied, all 26 subsequently stained TMA sections evaluated were of good/acceptable quality, and a high level of concordance in the assessment of the presence or absence of specific alphaS-IR structures was achieved. A semiquantitative assessment of alphaS-IR neuronal perikaryal inclusions yielded agreements ranging from 49% to 82%, with best concordance in cortical core samples. These results suggest that rigorous methodology and dichotomized assessment (i.e. determining the presence or absence of alphaS-IR) should be applied, and that semiquantitative assessment can be recommended only for the cortical samples. Moreover, the study demonstrates that there are limitations in the scoring of alphaS-IR structures.

Immunohistochemistry in the evaluation of neovascularization in tumor xenografts

Angiogenesis, or neovascularization, is known to play an important role in the neoplastic progression leading to metastasis. CD31 or Factor VIII-related antigen (F VIII RAg) immunohistochemistry is widely used in experimental studies for quantifying tumor neovascularization in immunocompromised animal models implanted with transformed human cell lines. Quantification, however, can be affected by variations in the methodology used to measure vascularization including antibody selection, antigen retrieval (AR) pretreatment, and evaluation techniques. To examine this further, we investigated the microvessel density (MVD) and the intensity of microvascular staining among five different human tumor xenografts and a mouse syngeneic tumor using anti-CD31 and F VIII RAg immunohistochemical staining. Different AR methods also were evaluated. Maximal retrieval of CD31 was achieved using 0.5 M Tris (pH 10) buffer, while maximum retrieval of F VIII RAg was achieved using 0.05% pepsin treatment of tissue sections. For each optimized retrieval condition, anti-CD31 highlighted small vessels better than F VIII RAg. Furthermore, the MVD of CD31 was significantly greater than that of F VIII RAg decorated vessels (p<0.001). The choice of antibody and AR method has a significant affect on immunohistochemical findings when studying angiogenesis. One also must use caution when comparing studies in the literature that use different techniques and reagents.

Cutaneous Syringoma: the Status of Hormonal Receptors

To the Editor: We read with great interest the report by Lee et al. on syringomas,1 and paid special attention to the immunohistochemical results obtained by the authors. All of the 56 cases that were studied for the expression of estrogen receptors (ER) or progesterone receptors (PR) were negative. As the authors admit, there are several previous reports in literature which corroborate the expression by syringoma of progesterone receptors.2-4 On the other hand, negativity has also previously been obtained in shorter series of syringomas,5-7 and even ourselves have seen negativity in an eruptive variant of syringoma that we reported.8 We wonder whether these apparently contradictory results might have something to do with the antigen-retrieval method used in each case. For instance,2 some authors have demonstrated the expression of ER and PR in syringoma, after retrieval with microwave for 10 minutes at 750 W with 10 mol/L citrate buffer at pH6, and most others teams used a wattage of around 800 W, when immunostaining for hormonal receptors.9 In the study by Lee et al., we regrettably failed to find the specification about the heating time of the samples or the wattage they used. Neither did they mention, if they used any controls. When several procedures for antigen retrieval have been compared in regard with the detection of hormonal receptors, pressure-cooking has proved to give the best results, and it is recommended for the standardization of examination of ER.10 In one study, 58% of laboratories that failed to assess immunohistochemistry for ER and PR, used microwave for antigen retrieval (compared to only 25% that failed by using the pressure cooker).9 Moreover, a short heating time was recognized as the main factor for poor immunohistochemical detection of ER and PR, in laboratories where microwave oven was used as an antigen-retrieval method.9 When extension of the heating time was introduced (up to total 25 minutes),9,11 significant improvement was achieved in the immunostaining for hormonal receptors, regardless of other variables in the study. Rhodes 2001 furthermore, it must be said that an increase of the heating time also improves those stains that are weak.9 Again, it would be interesting to know if the authors categorized any of their cases as negative, because of the extremely weak immunostaining for hormonal receptors. Those would be the cases where their staining could be improved by changing the retrieval conditions. We recently had an opportunity to check the evidence of ER and PR in four cases of syringomas, using the Dako REAL EnVision detection system, peroxidase/DAB+, rabbit/mouse as well as the pressure-cooking as an antigen-retrieval tool, at pH 9 for 2 minutes and 30 seconds. Endogen peroxidase was blocked with DakoCytomation peroxidase-blocking reagent (code S2021). We used the wash buffer DakoCytomation (code 53006) and progesterone receptor (Dakocytomation Clone PgR 636, Code N1630). We used primary antibodies for Estrogen receptor (Dakocytomation, monoclonal mouse antihuman, clone 1D5, code M7047) and progesterone receptor (Dakocytomation Clone PgR 636, Code N1630). Case 1 was a syringoma of the forehead in a 31-year-old female patient. Case 2 was a syringoma of the nose in a 44 year-old woman. Case 3 was a syringoma of the face in a 51 year-old-female patient. Case 4 presented as multiple papules in the thorax of a 13-year-old male with hypochondroplasia. None of the cases showed any expression of either ER or PR. Our results seem to support the findings of Lee at al., even when a pressure cooker was used as a retrieval tool, and our series is much shorter than theirs.

Microwave oven-based technique for immunofluorescent staining of paraffin-embedded tissues.

Immunohistochemical analysis of formalin-fixed paraffin-embedded tissues can be challenging due to potential modifications of protein structure by exposure to formalin. Heat-induced antigen retrieval techniques can reverse reactions between formalin and proteins that block antibody recognition. Interactions between antibodies and antigens are further enhanced by microwave irradiation, which has simplified immunohistochemical staining protocols. In this report, we modify a technique for antigen retrieval and immunofluorescent staining of formalin-fixed paraffin-embedded tissues by showing that it works well with several antibodies and buffers. This microwave-assisted method for antigen retrieval and immunofluorescent staining eliminates the need for blocking reagents and extended washes, which greatly simplifies the protocol allowing one to complete the analysis in less than 3 h.

PROTEIN EXPRESSION OF STEROIDOGENIC ENZYMES IN OVARIAN TUMORS OF THE DOMESTIC HEN

Advances in understanding the development of ovarian cancer may help in the identification of screening markers to detect early stages of the disease. Epidemiological data indicate that pregnancy and breast feeding are protective for ovarian cancer. The protective effects of reduced ovulations in these physiological states compared to the accompanying altered hormonal milieu, have been difficult to separate experimentally. Sex steroids have been implicated in ovarian cancer with evidence indicating that progesterone (P) inhibits the growth of tumor cells and that estrogen (E) promotes proliferation. Studies also suggest that ovarian tumors may be the site of synthesis of sex steroids. Like the human, the domestic hen spontaneously develops ovarian cancer in an age-related manner. We have previously found that 100% of ovarian tumors in the hen express ovalbumin, the major protein normally synthesized in the oviduct under the regulation of E. We have also demonstrated that hen tumors are positive for P and E receptors. In the present study, we examined the expression of 3-beta-hydroxysteroid dehydrogenase (3-beta- HSD) and aromatase in normal ovarian tissue and in ovarian tumors from the domestic hen. Tissues were fixed in formalin, embedded in paraffin, sectioned and mounted on glass slides. They were subsequently deparaffinized, rehydrated and processed using either of 2 antigen retrieval methods: boiling in citrate buffer for 20 minutes (for 3-beta-HSD antibody) or incubating in pronase for 30 minutes at 37° C (aromatase antibody). Primary antibody was applied for 16 hrs at 5° C and secondary antibody for 1 hr at 37° C. Tissues were examined with a Nikon E600 microscope under epifluorescence using fluorescein isothiocyanate excitation and barrier filter sets. Primary antibodies were rabbit polyclonal antiserum to recombinant human 3-beta-HSD diluted 1:1000, rabbit polyclonal antiserum to aromatase (Abcam, ab18995) used at a concentration of 20 μg/ml and IgG rabbit control serum (negative control). Secondary antibody was Alexa Fluor 488 goat anti-rabbit IgG conjugate (1.0 μg/ml; Invitrogen Molecular Probes). Ten of 14 (71%) ovarian tumors were positive for 3- beta-HSD protein with expression localized to the tubular/glandular areas of the tumors. In addition, expression was often observed in the theca or granulosa of normal follicles associated with the ovarian tumors. Follicles of normal ovaries (n=4) were positive for 3-beta-HSD and IgG controls (n=4) were negative. No ovarian tumors (n=10) were positive for aromatase in the tubular/glandular areas, however, 2 of the tumors had normal appearing follicles which were positive. Follicles of normal ovaries (n=4) were positive for aromatase and IgG controls (n=2) were negative. Our results indicate that 3-beta-HSD enzyme is present in the tubular/glandular areas of ovarian tumors and suggest that P or androgens could be synthesized in the tumors of the hen. Furthermore, aromatase activity is absent in the ovarian tumors of the hen with the exception of normal follicles and therefore, it is unlikely that E is being synthesized by the neoplastic cells. (poster)

Traumatic axonal damage in the brain can be detected using β‐APP immunohistochemistry within 35 min after head injury to human adults

Immunohistochemistry staining for beta-amyloid precursor protein (beta-APP) is a sensitive method to detect early axonal damage in traumatic brain injury, which was previously estimated to be of minimum 60-90 min after head injury. We present seven cases of well-documented posttraumatic survival of 35-60 min where beta-APP detects early axonal damage. Cases were selected from routine work where documentation about survival is judged to be accurate. These are divided into three groups: group 1: severe head injury (n = 7) with documented survival between 35 and 60 min. Group 2: severe head injury (n = 4) with documented survival of less than 30 min. Group 3: cases (n = 4) where death was not due to head injury but survival is documented between 45 and 109 min. The brains were fixed in formalin for 4 weeks and six regions (frontal lobe with anterior corpus callosum, parietal lobe with deep white matter, basal ganglia with posterior limb of internal capsule, cerebellum with white matter and middle cerebellar peduncle and pons with basis pontis and superior cerebellar peduncle) were sampled. All blocks were stained for haematoxylin and eosin and beta-APP and selected ones for CD68, using antigen retrieval method. In group 1 sections revealed beta-APP immunoreactivity in forms of small globules and granules and occasionally as thin and short filaments. These were detected in the pons, corpus callosum, internal capsule and cerebral white matter, with some variation in localization and intensity. In groups 2 and 3 all the sections were negative for beta-APP staining. None of the cases showed evidence of severe brain swelling, increased intracranial pressure, ischaemia or infection. Using the antigen retrieval method, beta-APP immunohistochemistry can detect axonal damage within 35 min after severe head injury. These results may have an implication in the consideration of minimal survival time after traumatic head injury in medico-legal practice.

Pepsin pretreatment allows collagen IV immunostaining of blood vessels in adult mouse brain

While the brain vasculature can be imaged with many methods, immunohistochemistry has distinct advantages due to its simplicity and applicability to archival tissue. However, immunohistochemical staining of the murine brain vasculature in aldehyde fixed tissue has proven elusive and inconsistent using current protocols. Here we investigated whether antigen retrieval methods could improve vascular staining in the adult mouse brain. We found that pepsin digestion prior to immunostaining unmasked widespread collagen IV staining of the cerebrovasculature in the adult mouse brain. Pepsin treatment also unmasked widespread vascular staining with laminin, but only marginally improved isolectin B4 staining and did not enhance vascular staining with fibronectin, perlecan or CD146. Collagen IV immunoperoxidase staining was easily combined with cresyl violet counterstaining making it suitable for stereological analyses of both vascular and neuronal parameters in the same tissue section. This method should be widely applicable for labeling the brain vasculature of the mouse in aldehyde fixed tissue from both normal and pathological states.

Glyoxal fixation: how it works and why it only occasionally needs antigen retrieval

Over the past 13 years, glyoxal has become the leading alternative to formaldehyde as a histological fixative because of its low inhalation risk, faster reaction rate and selective control over crosslinking. The latter attribute is especially important, because most of the difficulties relating to use of formaldehyde-fixed specimens for immunohistochemistry stem from its aggressive crosslinking behavior. With suitable catalysts or other reaction accelerators, glyoxal forms 2-carbon adducts with nearly all end groups in proteins and carbohydrates, leaving most of them unimpaired for subsequent immunohistochemical demonstration. Only arginine is seriously impaired by the formation of imidazoles, which is the basis for the well known arginine blockade method using glyoxal. A special glyoxal-specific antigen retrieval method using high pH and high temperature effectively reverses the blockade and restores immunoreactivity. Other methods for antigen retrieval are rarely beneficial and in most cases damage the specimen. Special stains work well, except silver methods for Helicobacter pylori. Routine hematoxylin and eosin preparations exhibit clarity and cellular detail rarely seen with formaldehyde.

Histochemistry for Placenta Research: Theory and Application

Histochemical techniques have contributed significantly to advances in placental biology and cell biology. In this mini-review, we describe recent advances in histochemical technologies and show how these technologies can profoundly improve our understanding of placenta morphological function related to health and disease. Fundamental theories and applications of five separate methods discussed here are 1) tissue-based polymerase chain reaction by laser microdissection, 2) a novel antigen retrieval method using citraconic anhydride plus heating, 3) immunohistochemical detection of Lewis-related antigen expression and galectin-1 binding in the human placenta, 4) confocal microscopy analysis of IgG transport in placental trophoblasts, and 5) high-resolution immunofluorescence and correlative microscopy using ultrathin cryosections in placental research. This review article is based on a presentation given in a workshop entitled Histochemistry: Theory and Application at the 12th International Federation of Placenta Associations Meeting held in Kobe, Japan, on September 9, 2006.

Practical methods for detecting melanocytes in the skin and their responses to UV

Recently, there has been significant literature and discussion about the effects of ultraviolet (UV) exposure on the human body. Pigmentation provided by melanin protects us from harmful effects of UV exposure (including skin cancer). Although keratinocytes are the most numerous type of cell in the skin, melanocytes are also key players since they produce and distribute melanin to protect the skin from UV radiation. In vitro experiments on melanocytic cell lines are helpful to study melanogenesis and the progression of melanocytes toward melanoma. However, interactions of melanocytes not only with keratinocytes but also with other types of cells in the skin, such as fibroblasts and Langerhans cells, also appear to be crucial. Using two techniques, immunohistochemistry and tissue in situ hybridization (TISH), we have optimized methods to identify and study melanocytes in the skin and their responses to UV in vivo. We describe a practical method to localize melanocytic antigens on formalin-fixed, paraffin-embedded tissue sections and frozen sections using indirect immunofluorescence with conjugated secondary antibodies. We also show a comparison of the effects of two methods of antigen retrieval to optimize staining of our melanocyte markers. In addition, we discuss the combination of immunohistochemistry with tissue in situ hybridization to the study mRNA levels of genes in the skin responding to UV. The importance of the TISH technique is underscored by the fact that it permits us to study expression of transcription factors involved in melanogenesis, to which antibodies are not available. Our methodology along with relevant tips and troubleshooting items are important tools in understanding melanocyte responses to UV.

The use of heat-induced hydrolysis in immunohistochemistry on angiotensin II (AT 1) receptors enhances the immunoreactivity in paraformaldehyde-fixed brain tissue of normotensive Sprague–Dawley rats

The research on components of the renin–angiotensin system delivered a broad image of angiotensin II-binding sites. Especially, immunohistochemistry (IHC) provided an exact anatomical localization of the AT 1 receptor in the rat brain. Yet, controversial results between in vitro receptor autoradiography and IHC as well as between immunohistochemical studies using various antisera started a vehement discussion concerning specificity and cross-reactivity of these antisera. In particular the magnocellular subdivision of the paraventricular nucleus (PVN) and the supraoptic nucleus (SON) provided controversial results on the localization of AT 1 receptors. Both areas are known for angiotensin II-induced release of vasopressin (VP) and oxytocin (OXT). To evaluate the significance of the appropriate method of antigen retrieval and its relevance for the detection of AT 1 receptors we performed IHC on AT 1 receptors in paraformaldehyde-fixed and paraffin-embedded brain tissue of Sprague–Dawley rats using either the detergent Triton X-100 or microwave oven heating. This study demonstrates that heat-induced hydrolysis enhances the quality and quantity of immunoreactivity (IR) in IHC on AT 1 receptors. In the organum vasculosum lamina terminalis and in the parvocellular subdivisions of the PVN we report a distribution of AT 1-like-IR similar to that observed with other methods. However, in addition, we provide evidence that distinct AT 1-like-IR is also localized in few magnocellular neurons of the PVN and in few parvocellular neurons of the dorsal SON but not in magnocellular neurons of the SON. Moreover, parallel IHC indicates that few magnocellular OXT- or VP-releasing neurons of the PVN as well as parvocellular OXT-releasing neurons of the SON do also contain AT 1 receptors.

Unmasking Hidden Epitopes with Proteases

INTRODUCTIONFixation can mask epitopes. However, it is often possible to re-expose them using a gentle incubation with proteases, which removes obstructing structures and allows antibody access, as described here. Many proteases can be used for this procedure, including very crude preparations of proteases, such as pronase. However, using a better-characterized protease, such as trypsin, allows a more controlled reaction and better comparison between experiments. Be aware that any of the antigen retrieval methods should be avoided wherever possible, because they may introduce artifactual false-positive staining. Methods for antigen retrieval are specific for each antibody/antigen combination. No attempts to establish quantitative data following these methods should be considered.

Unmasking Hidden Epitopes Using the Microwave Oven

INTRODUCTIONSeveral methods have been developed to "retrieve" antigens that have been masked by fixation. The principle behind using the microwave oven method described here is to use extended periods of heat to break some of the subcellular structures that block antibody access. Be aware that any of the antigen retrieval methods should be avoided wherever possible, because they may introduce artifactual false-positive staining. Methods for antigen retrieval are specific for each antibody/antigen combination. No attempts to establish quantitative data following these methods should be considered.

Unmasking Hidden Epitopes Using the Pressure Cooker

INTRODUCTIONSeveral methods have been developed to "retrieve" antigens that have been masked by fixation. The principle behind the pressure cooker method described here is to use extended periods of heat to break some of the subcellular structures that block antibody access. This approach is appropriate for handling specimens on glass slides. The major advantages of the pressure cooker method are the ability to handle a large number of slides simultaneously, the convenience of using metal racks, and the avoidance of any hot spots that are found in the microwave. Be aware that any of the antigen retrieval methods should be avoided wherever possible, because they may introduce artifactual false-positive staining. Methods for antigen retrieval are specific for each antibody/antigen combination. No attempts to establish quantitative data following these methods should be considered.