Aldehyde fuchsin staining of pancreatic B cells. Reproducible high-contrast staining of formalin-fixed and paraffin-embedded material

Summary The use of Aldehyde Fuchsin for the demonstration of B cell granules in the pancreatic islets on material fixed in formalin and embedded in paraffin has led to variable results.Treatment of such sections for 1 h with Bouin's fluid or 5% glutaraldehyde prior to deparaffinization, however, stabilizes the secretory granules in B cells. In addition, the zymogenic granules of the acinar cells exhibit increased stainability with the permanganate Aldehyde Fuchsin procedure.     

Combination of the peroxidase anti-peroxidase (PAP)-and avidin-biotin-peroxidase complex (ABC)-techniques: an amplification alternative in immunocytochemical staining

Summary A combination of the PAP- and ABC-techniques was developed to enhance the intensity of the immunocytochemical staining with monoclonal antibodies at light and electron microscopical levels. This amplification technique could be performed in 4 (single PAP + ABC) or 6 (double PAP + ABC) sequential steps depending on the quality of the primary antibodies used and the processing of the tissue before the immunocytochemical reaction: First step — Incubation of the tissue sections with the monoclonal primary antibodies; Second step — biotinylated anti-rat or anti-mouse IgG; Third step — monoclonal PAP complex; Fourth step — ABC complex which binds to the biotinylated secondary antibody. If stronger enhancement of the immunostaining has required the steps 2 and 3 could be repeated followed by the 6th step — the ABC complex. Choline acetyltransferase-like immunoreactivity of the rat hypoglossal nucleus and desmin- and vimentin-like immunoreactivity of human testis were studied. After the 4-and more pronounced the 6-step reaction a significant increase of the staining intensity was observed for all the reactions under study. ChAT-like immunoreactivity was observed to longer distances of the nerve cell dendrites after their emerging from the perikarya and within a greater number of structures in the neuropil as compared to the standard techniques. At electron microscopical level the technique permits longer fixation of the tissue which is important for the better preservation of the ultrastructure as well as for the easier recognition of the reaction product even in the smallest dendrite branches and the axons of the nerve cells. Stronger staining intensity was obtained for desmin-like immunoreactivity (LI) (within myofibroblasts of the lamina propria and muscle cells of the blood vessels)-and vimentin-LI (within Sertoli cells, myofibroblasts of the lamina propria and fibroblasts of the interstitium) of the human testis. The full combination (6 step-reaction) permits the detection of smallest quantities of an antigen in sections of different tissues using highly diluted primary antibodies. The technique represents a further alternative among the immunocytochemical amplification methods.     

Combination of the peroxidase anti-peroxidase (PAP)- and avidin-biotin-peroxidase complex (ABC)-techniques: an amplification alternative in immunocytochemical staining.

A combination of the PAP- and ABC-techniques was developed to enhance the intensity of the immunocytochemical staining with monoclonal antibodies at light and electron microscopical levels. This amplification technique could be performed in 4 (single PAP + ABC) or 6 (double PAP + ABC) sequential steps depending on the quality of the primary antibodies used and the processing of the tissue before the immunocytochemical reaction: First step--Incubation of the tissue sections with the monoclonal primary antibodies; Second step--biotinylated anti-rat or anti-mouse IgG; Third step--monoclonal PAP complex; Fourth step--ABC complex which binds to the biotinylated secondary antibody. If stronger enhancement of the immunostaining has required the steps 2 and 3 could be repeated followed by the 6th step--the ABC complex. Choline acetyltransferase-like immunoreactivity of the rat hypoglossal nucleus and desmin- and vimentin-like immunoreactivity of human testis were studied. After the 4- and more pronounced the 6-step reaction a significant increase of the staining intensity was observed for all the reactions under study. ChAT-like immunoreactivity was observed to longer distances of the nerve cell dendrites after their emerging from the perikarya and within a greater number of structures in the neuropil as compared to the standard techniques. At electron microscopical level the technique permits longer fixation of the tissue which is important for the better preservation of the ultrastructure as well as for the easier recognition of the reaction product even in the smallest dendrite branches and the axons of the nerve cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantitative imaging of immunocytochemical (PAP) estrogen receptor staining patterns in breast cancer sections

"Receptogram Analysis" has been developed as a pattern-oriented approach for predicting endocrine response in breast cancer based upon quantification of the estrogen receptor immunocytochemical assay (ERICA), using a Quantimet Imaging System. Response prediction was evaluated in 58 stage III and IV patients receiving endocrine therapy (primarily Tamoxifen). The Receptogram is a composite of the univariate distributions of nuclear receptor content, IOD(S), and concentration (MOD), and their bivariate contour plot; where (S) is the calculated nuclear radius in section. MOD distributions were classified into four types based upon peak modality and kurtosis (I-IV), and contour plots were classified into four subtypes (A-D) based upon contour slope. Patients failing therapy were ERICA--or their receptogram revealed co-existent ER+ and ER- tumor cells (type II), highly skewed MOD distributions lacking defined peaks (type IV), or contours with nearly horizontal slope (type C). Response was realized in 9/16 type I patients, with a single positive MOD peak, and in 9/15 type III patients, with discrete, multimodal MOD peaks. In contrast, 0/8 type II, 0/12 type IV, and 0/10 type C patients were responders. Receptogram analysis was superior to cytosol assay (DCC) as a response discriminant: positive predictive value, 53% vs. 33%; negative predictive value, 100% vs. 75%; sensitivity, 100% vs. 83%; specificity, 68% vs. 23%; and accuracy, 78% vs. 41%, respectively. Alternately, patients were assigned to potentially responsive or non-responsive groups based upon thresholded mean receptor parameters: field MOD, mean nuclear MOD (NMOD), and mean NMOD(PF) where PF is the ER+ nuclear fraction. While these parameters correlated with DCC (r = .72, 0.69, and 0.69), they were only marginally better in predictive value.     

The use of proteolytic enzymes to improve immunoglobulin staining by the PAP technique

Synopsis Proteolytic enzymes, protease and trypsin have recently been introduced to reduce the inconsistency hitherto encountered in the unlabelled antibody-enzyme method using PAP. This study investigated factors determining the optimum conditions for use of such enzymes in order to establish which one is most suitable. Trypsin was the most effective enzyme; however, its activity decreased over 3 h, a feature paralleled immunocytochemically. Method and duration of fixation appears to influence the required time of exposure to trypsin in order that consistent immunostaining may be produced. Treatment of sections with trypsin prior to the use of the unlabelled antibody-enzyme method using PAP renders the technique reliable, provided the enzyme is used in a carefully controlled manner.     

Identification of methods for use of formalin-fixed, paraffin-embedded tissue samples in RNA expression profiling

Formalin-fixed paraffin-embedded (FFPE) tissue samples are a potentially valuable resource of expression information for medical research, but are under-utilized due to degradation and modification of the RNA. Using a random primer-based RNA amplification strategy, we have evaluated multiple protocols for the extraction and isolation of RNA from FFPE samples. We found that the RecoverAll RNA isolation procedure with three or four slices (ten-microns in thickness), supplemented with additional DNAse, gave optimal results. RNA integrity as assessed by Agilent Bioanalyzer, and amplification of the 28S ribosomal RNA, were predictive for the number of genes detected on Affymetrix arrays. We obtained expression data for colon and lung tumor and normal FFPE samples and matched frozen samples and found a high correlation between frozen and matched FFPE samples (R² between 0.82 and 0.89), while the signature sets in tumor versus normal comparisons were also quite similar. QPCR confirmed all 16 of the differential expression results from the microarrays that we tested. Differentially expressed signature genes from tumor versus matched normal FFPE tissue from colon and lung were identified as cancer-related, with 95 colon tumor and 67 lung tumor genes identified, respectively.     

Proteomic analysis of formalin-fixed paraffin-embedded tissue by MALDI imaging mass spectrometry

Archived formalin-fixed paraffin-embedded (FFPE) tissue collections represent a valuable informational resource for proteomic studies. Multiple FFPE core biopsies can be assembled in a single block to form tissue microarrays (TMAs). We describe a protocol for analyzing protein in FFPE-TMAs using matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS). The workflow incorporates an antigen retrieval step following deparaffinization, in situ trypsin digestion, matrix application and then mass spectrometry signal acquisition. The direct analysis of FFPE-TMA tissue using IMS allows direct analysis of multiple tissue samples in a single experiment without extraction and purification of proteins. The advantages of high speed and throughput, easy sample handling and excellent reproducibility make this technology a favorable approach for the proteomic analysis of clinical research cohorts with large sample numbers. For example, TMA analysis of 300 FFPE cores would typically require 6 h of total time through data acquisition, not including data analysis.     

p53 overexpression in formalin-fixed, paraffin-embedded tissue detected by immunohistochemistry.

Mutation and overexpression of the p53 gene have been noted in a wide range of human cancers and are thought to play a role in malignant transformation. Previously, immunohistochemical detection of p53 has been possible only in fresh-frozen tissues. We examined p53 expression in paraffin-embedded tissues from 50 epithelial ovarian cancers and 25 primary breast cancers with a modified immunohistochemical (IHC) technique developed in this laboratory, using monoclonal antibody (MAb) PAb1801. The 75 cases were selected from a group of patients in whom the expression levels had already been assessed in a fresh-tissue IHC assay. An identical staining reactivity was observed in both formalin-fixed, paraffin-embedded tissue and fresh-frozen tissue in 48 of 50 (96%) epithelial ovarian cancers and in 23 of 25 (92%) primary breast cancers. Immunodetection of p53 in paraffin-embedded tissue blocks will be a useful alternative to the standard fresh-tissue assay and can accurately reflect the level of p53 expression in human tumors.     

Proteome analysis of microdissected formalin-fixed and paraffin-embedded tissue specimens.

Targeted proteomics research, based on the enrichment of disease-relevant proteins from isolated cell populations selected from high-quality tissue specimens, offers great potential for the identification of diagnostic, prognostic, and predictive biological markers for use in the clinical setting and during preclinical testing and clinical trials, as well as for the discovery and validation of new protein drug targets. Formalin-fixed and paraffin-embedded (FFPE) tissue collections, with attached clinical and outcome information, are invaluable resources for conducting retrospective protein biomarker investigations and performing translational studies of cancer and other diseases. Combined capillary isoelectric focusing/nano-reversed-phase liquid chromatography separations equipped with nano-electrospray ionization-tandem mass spectrometry are employed for the studies of proteins extracted from microdissected FFPE glioblastoma tissues using a heat-induced antigen retrieval (AR) technique. A total of 14,478 distinct peptides are identified, leading to the identification of 2733 non-redundant SwissProt protein entries. Eighty-three percent of identified FFPE tissue proteins overlap with those obtained from the pellet fraction of fresh-frozen tissue of the same patient. This large degree of protein overlapping is attributed to the application of detergent-based protein extraction in both the cell pellet preparation protocol and the AR technique.     

C4d immunohistochemical staining is a sensitive method to confirm immunoreactant deposition in formalin-fixed paraffin-embedded tissue in membranous glomerulonephritis

Although the diagnosis of membranous glomerulonephritis (MGN) may be suspected on routine histology of formalin-fixed paraffin-embedded tissue, fresh-frozen tissue must be used to show the immunologic nature of the process by direct immunofluorescence (IF). The efficiency of IF or immunoperoxidase (IP) detection of IgG and C3 using paraffin sections is controversial. This study was designed to evaluate whether glomerular C4d deposition using an IP method in formalin-fixed paraffin-embedded tissue may be a useful marker for MGN. We showed characteristic glomerular, granular basement membrane deposition of C4d in 31 (100%) cases of idiopathic MGN and in 5 cases (100%) of pure class V membranous lupus nephritis, in which we had a positive diagnosis of the lesions for conventional IF study. Control cases were negative. Nineteen cases of different glomerulopathies, including IgA nephropathy, primary type I membranoproliferative glomerulonephritis, focal segmental glomerulosclerosis and minimal change disease showed diverse reproducible patterns of C4d deposition, without intrinsic background. Our results indicate that staining of formalin-fixed paraffin-embedded tissue for C4d can be used for confirmation of granular basement membrane immunoreactant deposition in cases of MGN. This proved to be a reliable method that could potentially obviate the need for rebiopsy in cases with absence of glomeruli in renal frozen sections or when other adjunct IF or IP methods on paraffin sections are negative. C4d immunostaining, using an IP method, deserves a place as an adjunct method in the biopsy diagnosis of MGN.     

Antigen retrieval for proteomic characterization of formalin-fixed and paraffin-embedded tissues

Formalin-fixed and paraffin-embedded tissues represent the vast majority of archived tissue. Access to such tissue specimens via shotgun-based proteomic analyses may open new avenues for both prospective and retrospective translational research. In this study, we evaluate the effects of fixation time on antigen retrieval for the purposes of shotgun proteomics. For the first time, we demonstrate the capability of a capillary isotachophoresis (CITP)-based proteomic platform for the shotgun proteomic analysis of proteins recovered from FFPE tissues. In comparison to our previous studies utilizing capillary isoelectric focusing, the CITP-based analysis is more robust and increases proteome coverage. In this case, results from three FFPE liver tissues yield a total of 4098 distinct Swiss-Prot identifications at a 1% false-discovery rate. To judge the accuracy of these assignments, immunohistochemistry is performed on a panel of 17 commonly assayed proteins. These proteins span a wide range of protein abundances as inferred from relative quantitation via spectral counting. Among the panel were 4 proteins identified by a single peptide hit, including three clusters of differentiation (CD) markers: CD74, CD117, and CD45. Because single peptide hits are often regarded with skepticism, it is notable that all proteins tested by IHC stained positive.     

Differential staining of nerve cells and fibres for sections of paraffin-embedded material in mammalian central nervous system

A differential staining method is described of myelinated fibres and nerve cell bodies applicable to sections of mammalian, including human, central nervous system specimens embedded in paraffin wax. Experimental and human necropsy material fixed in acetic paraformaldehyde in phosphate buffer was used. Sections of 15–20 m in thickness were obtained, attached to slides, deparaffinized and hydrated. After hydration, sections are oxidized (30 s) in 2% potassium permanganate, bleached (1 min) in 5% oxalic acid and rinsed in distilled water. Staining is for 2–5 h in the following solution: 0.06% thionin, 1% formaldehyde, 10% acetic acid in distilled water. Sections are subsequently washed in distilled water, dehydrated through 96% and absolute ethanol, cleared in eucalyptol and mounted in Eukitt. Using the method described in the present paper, a differential coloration of myelin and neurons is obtained. Myelinated fibres appear red, whereas nerve cell bodies and glial nuclei are stained blue. This procedure provides a high contrast between myelin and cells suitable for observation and photography of sections. Simultaneous and differential coloration of both myelin and cells is easily and directly obtained with constant and homogeneous results.     

Direct analysis and MALDI imaging of formalin-fixed, paraffin-embedded tissue sections

Formalin fixation, generally followed by paraffin embedding, is the standard and well-established processing method employed by pathologist. This treatment conserves and stabilizes biopsy samples for years. Analysis of FFPE tissues from biopsy libraries has been, so far, a challenge for proteomics biomarker studies. Herein, we present two methods for the direct analysis of formalin-fixed, paraffin-embedded (FFPE) tissues by MALDI-MS. The first is based on the use of a reactive matrix, 2,4-dinitrophenylhydrazine, useful for FFPE tissues stored less than 1 year. The second approach is applicable for all FFPE tissues regardless of conservation time. The strategy is based on in situ enzymatic digestion of the tissue section after paraffin removal. In situ digestion can be performed on a specific area of the tissue as well as on a very small area (microdigestion). Combining automated microdigestion of a predefined tissue array with either in situ extraction prior to classical nanoLC/MS-MS analysis or automated microspotting of MALDI matrix according to the same array allows the identification of both proteins by nanoLC-nanoESI and MALDI imaging. When adjacent tissue sections are used, it is, thus, possible to correlate protein identification and molecular imaging. These combined approaches, along with FFPE tissue analysis provide access to massive amounts of archived samples in the clinical pathology setting.     

Expression analysis of mRNA in formalin-fixed, paraffin-embedded archival tissues by mRNA in situ hybridization

Gene expression in diseased tissues can indicate the contribution to a disease process and potentially guide therapeutic decision-making. Archival tissues with associated clinical outcome may be useful to discover or validate the role of a candidate gene in a disease process or the response to therapy. Such archival tissues are commonly formalin-fixed and paraffin-embedded, restricting the methods available for gene expression analysis. Obviously, the detection of proteins in tissues requires adaptation for each protein and the detection of secreted proteins can prove difficult or of reduced value since the protein detected may not reflect the total amount produced. Thus, we describe here a reliable method for the detection of mRNA in archival tissues. The method for mRNA in situ hybridization (ISH) was adapted by us for >15 different genes and applied to several hundred tissue microarrays (TMAs) and full sections generating >10,000 expression data points. We also discuss the utility of TMAs to simultaneously analyze several hundred tissue samples on one slide to minimize variability and preserve valuable tissue samples. Experimental protocols are provided that can be implemented without major hurdles in a typical molecular pathology laboratory and we discuss quantitative analysis as well as advantages and limitations of ISH with a special focus on secreted proteins. We conclude that ISH is a reliable and cost effective approach to gene expression analysis in archival tissues that is amenable to screening of series of tissues or of genes of interest.