Immunoperoxidase study of lymphomas. Comparison of a one-step frozen section technic with indirect methods on paraffin sections

Fifty cases of non-Hodgkin's lymphoma (15 nodular and 35 diffuse) were studied to determine the sensitivity, specificity, and ease of several different immunoperoxidase methods. The methods included a rapid, simple one-step immunoperoxidase procedure on frozen sections compared with indirect immunoperoxidase technics on paraffin sections. The frozen-section immunoperoxidase technic stained 15 of 15 nodular lymphomas and 24 of 35 diffuse lymphomas for monoclonal light chain. The majority of the diffuse lymphomas that did not stain for light chains were morphologically and immunohistochemically consistent with T-cell lymphomas. The indirect method on B-5 and formalin-fixed tissues only rarely displayed monoclonal staining for nonplasmacytoid small cell lymphomas but did stain some large cell lymphomas and a majority of plasmacytoid lymphomas for monoclonal light chain. The frozen section technic presented in this report is sufficiently sensitive and reliable to detect immunoglobulins in any morphologic subtype of B-cell lymphoma, whereas paraffin-embedded tissues have only limited application.     

The immunogold-silver staining method

Summary Immunostaining of routinely fixed, wax embedded tissues may present problems to the pathologist since destruction of antigens can lead to false negative results. In an attempt to overcome this problem, we have compared the results of the standard peroxidase anti-peroxidase (PAP) method with those obtained using the newly developed and very sensitive immunogold-silver staining (IGSS) method. Sections from routine histopathological material as well as from normal tissue specimens were used in the comparison. Antisera to a variety of antigens commonly employed in pathology were used, including regulatory peptides and a range of other markers. In all cases the IGSS method was found to give superior or at least equal results to those obtained with the PAP technique. In some cases staining was obtained with IGSS method when the PAP technique gave no result. The intense black reaction product allowed much easier and more rapid screening of immunostained preparations as well as permitting sections to be counterstained with routine histological stains such as haematoxylin and eosin.It is therefore suggested that immunogold-silver staining is a valuable technique for the pathologist, particularly when examining overfixed or badly processed tissues.     

Immunocytochemical staining of progesterone receptor in paraffin sections of human breast cancers.

Monoclonal antibodies (mAB) against progesterone receptor (PR) and the peroxidase-antiperoxidase (PAP) method to visualize PR in paraffin sections from 68 human breast cancers were used. Ten mAB, which recognize human PR on frozen sections, were tested. Six could detect PR in paraffin sections, with Li 417, LET 456, and LET 126 giving the best results. LET 126 antibody was used for most further studies. The effects of fixation with picric acid-formaldehyde (PAF), buffered-formalin, or with Bouin solution were investigated; all fixation methods allowed PR immunolabeling, although PAF or buffered formalin usually gave the best results. Positive staining was seen in the nucleus of carcinoma cells. Variations in intensity and extent of immunoreactivity were observed in all sections and among different regions of the same specimen. These were probably related to the heterogeneity of the tumor cell population. Results were compared with the PR content in the respective tumor tissues, determined by steroid-binding assay, and with immunocytochemistry on frozen sections. It was shown that there were correlations between the immunocytochemical staining (positive or negative) and the steroid binding assay (80%) and between the immunocytochemical staining on paraffin sections and on frozen sections (78%). Weaker intensity and fewer number of PR-positive cells were found for paraffin-embedded tumors. Estrogen receptors were also detected on adjacent sections from the same paraffin-embedded tissues by use of monoclonal anti-ER antibodies (ERICA-kit[Abbott Laboratories, Chicago, IL]) and DNase pretreatment. In conclusion, this immunocytochemical method for detection of PR and ER on paraffin sections offers an alternative to frozen tissue. It allows histologic and immunocytochemical studies on the same sample and retrospective studies on stored tissue blocks.     

Immunohistologic analysis of lymphoid cells by a rapid double staining method

The development of monoclonal antibodies has allowed characterization of subpopulations of lymphoid cells and of their in situ distribution in tissues. The feasibility of simultaneous localization of two surface antigens was studied by double staining with monoclonal antibodies to B cells, T cell subsets and follicular dendritic reticulum cells (DRC) using the avidin-biotin-complex (ABC) method in sections of human lymphoid tissues (tonsil, lymph node, spleen) and a non-lymphoid tissue, endometrium. Color mixture was avoided when an additional incubation with avidin-biotin-labeled peroxidase and subsequent development in the respective substrate of the first sequence preceded the second staining sequence using the primary antibodies at optimal concentrations. The antigenic profiles were portrayed by contrasting and distinct colors of the reaction products. It was observed that T lymphocytes of the cytotoxic/suppressor and helper/inducer phenotypes were topographically associated with each other and with B cells in B and T cell areas of lymphoid tissues as well as in lymphocytic aggregates in endometria. Subpopulations of these cells were mantled by processes of DRCs in lymphoid follicles. The findings indicate that the double ABC staining method can be used for simultaneous demonstration of two surface antigens in tissue sections.     

Application of immunogold-silver staining and immunoenzymatic methods in multiple labelling of human pancreatic Langerhans islet cells

The use of immunogold-silver staining (IGSS) combined with immunoperoxidase and/or immunoalkaline phosphatase methods for the simultaneous demonstration of pancreatic islet cell hormones on routinely fixed paraffin-embedded human tissue sections was examined. If IGSS was applied first, the black colour of silver-enhanced colloidal gold on doubly immunostained sections contrasted with the colours of most of the chromogens used generally in the 2 immunoenzymatic methods. If IGSS was followed by immunoalkaline phosphatase and immunoperoxidase techniques in optional sequence, 3 different hormone-containing cell types could be stained simultaneously without non-specific cross-reactions. IGSS and immunoalkaline phosphatase methods, together with 2 kinds of non-cross-reacting immunoperoxidase systems, permitted the detection of 4 distinct antigens on the same tissue section. Multiple immunohistochemical labelling of the endocrine pancreas provides an opportunity for the correct and rapid analysis of the topographic and morphometric relationships between different hormone-producing cell populations under both normal and pathological conditions. IGSS is of great potential for the simultaneous immunolabelling of antigens situated within separate cells.     

Silver enhancement of polymerised diaminobenzidine: increased sensitivity for immunoperoxidase staining

Unambiguous identification of lymphocytes is sometimes difficult because of weak immunostaining of the cell membrane immunoglobulins. A simple method of intensifying the diaminobenzidine (DAB) peroxidase reaction was therefore devised. Paraffin wax sections of formalin fixed tonsils and lymphomas were digested with trypsin and immunostained for kappa and lambda light immunoglobulin chains and CD3 antigen by various peroxidase linked detection systems. After reaction with hydrogen peroxide and DAB the sections were immersed in methenamine silver solution at 60 degrees C for three to seven minutes. The light brown stain on the cell membranes of the mantle zone lymphocytes became dark brown and the stronger stain of the plasma cells became black. Mantle zone B lymphocytes and CD3 positive T lymphocytes were precisely outlined even at low magnification and the lymphomas were easily classified as monoclonal or polyclonal. At high magnification, staining was clearer than with the immunogold-silver stain. Cryostat and paraffin wax sections of other tissues immunostained for various antigens showed similar intensification. Silver methenamine provides an easy means of increasing the sensitivity and visual impact of an immunoperoxidase/DAB reaction in any preparation.     

Immunohistochemical demonstration of c-erbB-2 oncoprotein in gastric adenocarcinoma: comparison of cryostat and paraffin wax sections and effect of fixation.

AIMS--To investigate the effects of fixation on the immunohistochemical demonstration of c-erbB-2 oncoprotein using paraffin wax and cryostat sections; to compare c-erbB-2 expression in non-neoplastic and neoplastic gastric tissues. METHODS--Adjacent blocks of tumour and non-neoplastic tissue from four gastrectomy specimens were put into a panel of 10 fixatives including acetone, B5, Bouin's fluid, Carnoy's fluid, buffered formalin, formol dichromate, zinc formalin, 4% paraformaldehyde, periodate-lysine-paraformaldehyde (PLP) and periodate-lysine-paraformaldehyde-dichromate (PLPD) before embedding in paraffin wax for sectioning. Similar tissue blocks were snap frozen and cryostat sections were postfixed in these fixatives, either alone or in combination, before immunostaining. RESULTS--In paraffin wax embedded sections the best fixative was PLP, and in frozen tissues the best results were obtained after fixation of cryostat sections in buffered formalin followed by cold methanol and acetone. Applying these fixatives to samples from a further 16 gastrectomy specimens, strong membrane staining of c-erbB-2 protein was found in the tumour in eight of 16 cases (50%) using paraffin wax sections, and staining was stronger in the better differentiated carcinomas. For frozen tissues, positive membrane staining was found in all gastric adenocarcinomas, but differential staining intensity associated with tumour differentiation could not be detected. CONCLUSIONS--These results indicate that fixation and paraffin wax embedding affect the results of immunohistochemical demonstration of c-erbB-2 in gastric cancer. The choice of fixative is critical in the demonstration and evaluation of c-erbB-2 protein expression by immunohistochemistry in gastric carcinomas. Staining results also vary depending on whether frozen or paraffin wax embedded tissues are studied.     

Identification of monoclonal antibodies to pancreatic islet cells by immunoperoxidase staining

Immunoperoxidase staining and enzyme-linked immunosorbent assay (ELISA) were used to identify monoclonal antibodies that reacted with pancreatic islet cells. All monoclonal antibodies produced against isolated human or rat pancreatic islets including one mouse autoantibody reacted with pancreatic islets in formalin-fixed pancreas sections, but not with rat kidney or thyroid. Reactivity was also found with suspensions of normal rat islet cells and rat insulinoma cells using a 3-stage immunoperoxidase procedure and an ELISA technique. Differences were observed in staining intensity between the various antigenic substrates tested suggesting variable cross-reactivity and/or number of epitopes. The sensitivity of the immunoperoxidase technique proved to be favourable for identification of monoclonal antibodies that recognize cellular constituents such as islet cell antigens present in low concentrations.     

Leucocyte antigens in human post mortem tissues: their preservation and loss as demonstrated by monoclonal antibody immunohistological staining

The development of monoclonal antibodies and of techniques for demonstrating antigens in situ in frozen tissue sections has been responsible for remarkable progress in diagnostic histopathology. We explored the potential of these techniques when applied to post mortem tissues that were frozen at various intervals after death and stained by monoclonal antibodies using immunoperoxidase technique. The monoclonal antibodies were selected according to their reactivity with essential markers of the lymphatic system and also to their availability. Lymph nodes and splenic tissue from 30 autopsy cases were stained in addition to thymic tissue from eight deceased infants. The antigens proved to be surprisingly well preserved. Staining could be precisely evaluated with UCHT1, OKT6 and anti-Leu-7 antibodies at least 72 h after death; staining with DAKO-pan-B, DAKO-LC and anti-Leu-3a was also very reliable. Antigens expressed by T-suppressor lymphocytes and dendritic reticulum cells were less well preserved. The T8-antigen of suppressor lymphocytes was usually demonstrable in the lymph nodes but less frequently in the splenic tissues. It is concluded that most leucocyte antigens are very resistant to post mortem disintegration, and that they can be reliably interpreted by immunohistological staining using monoclonal antibodies. We therefore recommend this in autopsy specimens in cases where in-vivo examination was not feasible.     

Application of immunofluorescent staining on paraffin sections improved by trypsin digestion.

The purpose of this study was to explore a method by which an improved immunofluorescent staining can be applied to formalin-fixed paraffin sections to demonstrate cellular or tissue deposits of immunoglobulins, complement and fibrin, and to demonstrate alpha-1-antitrypsin storage and hepatitis B antigens in liver, toxoplasma in heart, and carcinoembryonic antigens in colonic cancer. It was shown that immunohistochemical demonstration for the above mentioned antigens, but not for complement, was feasible. The paraffin sections were first treated with trypsin and the indirect staining method was used. The trypsin treatment was found to decrease the nonspecific background fluorescence through digestion of the tissue. It probably also unmasked the immunoreactive sites of viral antigens and alpha-1-antitrypsin. In general, a 2-hour digestion was satisfactory for the types of tissues examined in this study, and an optimal period of digestion could be sought to obtain the best result for a specific antigen. This method may be a useful adjuvant to histopathologic study, in which a retrospective immunohistochemical examination may be desirable.     

Immunocytochemical demonstration of astrocytes in brain sections combined with Nissl staining

The aim of the present study was to develop an easy and reliable protocol of combined preparation staining, which would unite the advantages of immunocytochemical demonstration of astrocytes with the availability to evaluate functional state of neurons provided by Nissl technique. The presented protocol of paraffin sections processing allows to retain high quality of tissue structure and provides for selective demonstration of astrocytes using the monoclonal antibodies against glial fibrillary acidic protein and contrast Nissl staining of cells. The protocol can be used without any changes for processing of brain sections obtained from the humans and other mammals with the exception of mice and rabbits.     

Comparative study for the detection of peritubular capillary C4d deposition in human renal allografts using different methodologies

Detection of peritubular capillary (PTC) C4d deposition in tissue sections of renal allograft biopsies became an important aid in the diagnosis of antibody-mediated rejection. Pathologists in many major transplant centers now routinely stain renal allograft biopsies for C4d. Currently, there are 3 commercially available antibodies. Two of these antibodies are monoclonal and are usually used with either a 3- or a 2-step indirect immunofluorescence (IF) methodology on frozen sections. A polyclonal antibody is used on formalin-fixed, paraffin-embedded tissue section with an immunoperoxidase detection system. The goal of our study was to compare these antibodies and methodologies in our renal allograft biopsy material. Twenty renal allograft biopsies with diffuse or focal PTC C4d staining, using immunofluorescence methods on frozen sections, were selected for this study. These biopsies were tested with the 3 commercially available anti-C4d antibodies (Biogenesis, Brentwood, Calif, cat no. 222-8004; Quidel Corporation, Santa Clara, Calif, cat no. A213; and ALPCO Diagnostic, Windham, NH, cat no. 004-BI-RC4D). Both monoclonal antibodies (Biogenesis and Quidel) were tested with a 3- and a 2-step indirect IF method on frozen sections. The polyclonal antibody (ALPCO) was applied to formalin-fixed paraffin sections using immunoperoxidase methodology. In selected cases, the polyclonal antibody was tested on frozen sections with a 3-step indirect IF method. To exclude possible false-negative staining with the IF method, we selected 10 additional biopsies that showed PTC margination of inflammatory cells, but were C4d-negative or only focally positive, and tested them with the ALPCO antibody on paraffin sections. We have found that all methodologies and antibodies tested provided adequate results with only minor differences between them. Perhaps the most sensitive method is the 3-step indirect IF on frozen sections using one of the monoclonal antibodies. We prefer the 2-step indirect IF method with the Quidel monoclonal antibody because of its simplicity, quick turnaround time, and relatively low cost. The advantages and disadvantages of the individual methodologies are discussed.     

Production of a monoclonal antibody as immunohistochemical marker on paraffin embedded tissues using a new immunization method

This report describes a method for the production of murine monoclonal antibodies (MAbs) against cellular antigens preserved during formol fixation and paraffin embedding of human tissues in an attempt to select markers that would be useful in immunopathology. Hybridomas were prepared using spleen cells from mice immunized with cell suspensions obtained from formalin-fixed paraffin block sections of a human breast carcinoma. A monoclonal antibody 83 D4 was selected, which was reactive with paraffin embedded breast carcinoma tissues, but not with normal breast. The reactive antigen has a high molecular weight (400-1000 kD) and was detected on the cell surface of live human breast cancer cell lines and on frozen tissues sections. These results demonstrate that the MAb 83 D4 identifies a native breast tumor associated epitope conserved during tissue fixation and embedding and could be used as an immunohistochemical marker.     

An immunocytochemical method for demonstrating estrogen receptor in human uterus using monoclonal antibodies to human estrophilin

We have used monoclonal antiestrophilin antibodies to develop an improved immunocytochemical method for localizing estrogen receptors in tissue sections with an indirect immunoperoxidase technique. Rat monoclonal antibodies were raised against human estrogen receptor (estrophilin) protein derived from the cytosol of MCF-7 human breast cancer cells. These monoclonal antibodies have been shown to have extensive cross-reactivity with estrogen receptors from various primate and nonprimate tissues. We have used frozen sections of human proliferative phase endometrium and an indirect immunoperoxidase technique to establish conditions for demonstrating estrogen receptor antigenic determinants in frozen tissue sections. The method involves (a) brief fixation with formaldehyde-containing fixatives either prior to freezing or immediately after cutting cryostat sections, (b) bleaching the tissue of endogenous peroxidase activity, (c) application of primary antibody or control immunoglobulin, (d) application of an adsorbed bridging antibody (goat antirat IgG), and (e) application of rat peroxidase-antiperoxidase followed by diaminobenzidine. Specific nuclear staining for estrogen receptor antigenic determinants was observed in the vast majority of epithelial and stromal cells. No specific cytoplasmic staining was identified in cryostat sections of any of the 17 cases studied for this report.     

The demonstration of B-cell, T-cell and myeloid antigens in paraffin sections

The monoclonal antibodies MB1 and MT1, which detect B cells and T cells respectively, have been applied to human lymphoid tissues. The distribution of staining within paraffin sections was compared with that observed using frozen sections and was found to be identical. The antibodies were then applied to paraffin sections of 19 B-cell lymphomas and 10 T-cell lesions in which full immunophenotyping had been performed. The B lymphomas all consisted of a large majority of MB1 positive cells with a variable infiltrate of small MT1 positive lymphocytes. The T cell lesions consisted of MT1 positive cells with few MB1 positive cells except in residual B cell areas of lymph nodes. In paraffin sections from cases of Hodgkin's disease anti-Leu M1 identified Reed-Sternberg cells and their variants and MB1 and MT1 showed a similar distribution of B cells and T cells to that demonstrated in previous studies using frozen sections. The results show that MB1 and MT1 are useful markers for B and T cells in routinely fixed paraffin embedded tissue. In conjunction with anti-Leu M1 they provide a valuable panel of antisera for the examination of lymph nodes and other biopsies when frozen tissue is not available.     

Use of freeze-dried paraffin-embedded sections for immunohistologic staining with monoclonal antibodies

Most diagnostically valuable monoclonal antibodies recognize antigens that do not survive conventional tissue processing. The use of frozen tissue sections for immunohistologic studies overcomes this obstacle but introduces a number of practical problems, e.g., the necessity to store material in the frozen state, poor morphologic preservation, etc. In the present paper we report that antigenic denaturation during conventional tissue processing appears to occur during exposure to aldehyde-containing fixatives and to alcohol but not as a result of heating or exposure to melted paraffin wax. In consequence, we have developed a technique by which tissue is freeze-dried and then embedded directly in paraffin wax. All but one of the 40 monoclonal antibodies investigated stained the freeze-dried paraffin sections with an intensity equal to or greater than that observed on frozen sections. There was less diffusion artifact and less background staining than in cryostat sections, and cellular morphology was better preserved. One of the most important advantages of this new method is that antigens in freeze-dried paraffin-embedded tissue are stable, and tissue blocks may be handled in the same manner as conventional paraffin blocks. An additional finding was that, once the tissue has been freeze-dried, paraffin embedded, and sectioned, the antigens it contains are resistant to fixatives (e.g., formol, formol sublimate, alcohols) which would very rapidly cause their destruction in frozen sections.     

Silver Acetate Autometallography: An Alternative Enhancement Technique for Immunogold-Silver Staining (IGSS) and Silver Amplification of Gold,Silver, Mercury and Zinc in Tissues

Abstract

Silver lactate autometallography in immunogold silver staining (IGSS) usually requires development in darkness to avoid excessive background staining. Our alternative method of silver enhancement of IGSS on paraffin sections from Bouin's or formalin fixed pancreas uses silver acetate in combination with hydroquinone in low pH buffer. The modification was tested with a range of antibodies in normal and diseased tissues (all routinely fixed and paraffin embedded), in acetone postfixed cryostat sections, and in semithin sections of glutaraldehyde fixed and resin embedded tissues. Silver acetate autometallography was also tested in various systems for the visualization of tissue metals like sulfides and selenides of mercury and zinc, silver, and gold. Comparisons between sections exposed to silver lactate and the silver acetate developer showed no significant difference in the number of structures stained. The degree of background staining was often lower when silver acetate was to used as the ion donor, especially with IGSS. The advantage the technique described here is that the development process can be controlled, using normal bright field light microscopy. (The J Histotechnol 11:213, 1988.)     

The immunohistochemical demonstration of major histocompatibility antigens in the human kidney using monoclonal antibodies

The distribution of HLA-ABC and DR antigens in human kidneys was studied using monoclonal antibodies to monomorphic determinants and a 4-layer immunoperoxidase technique applied to frozen and paraffin sections. Enzyme digested paraffin sections provided the best localization. HLA-ABC antigens were located on all renal endothelium, Bowman's capsule and in proximal tubules. HLA-DR antigen was restricted to capillary and venous endothelium plus, at low density, in proximal tubules. Minor differences in distribution were found with different monoclonal antibodies, but dendritic cells were not detected using monoclonal antibodies to HLA-ABC, HLA-DR or leukocyte-common antigens.     

Localization of Blood-Brain Barrier-Specific Antibodies with Immunogold-Silver Enhancement

Abstract

Epithelial-like tight junctions in vertebrate brain capillary endothelium constitute the blood-brain barrier (BBB). The function of the BBB is in part regulated by proteins that are selectively expressed in brain capillary endothelium, and the function of brain capillary specific proteins may be elucidated with immunogold-silver staining (IGSS) techniques and with antibodies specific to BBB proteins. Three endothelial enriched proteins are described in the present studies: the GLUT1 glucose transporter isoform, the endothelial transferring receptor, and immunoreactive brain capillary specific proteins (BSPs). Three different rabbit or mouse antibodies were used. At the light microscope level, IGSS techniques are as sensitive as avidin-biotin immunoperoxidase histochemistry. The use of gold conjugated to monoclonal antibodies specific to the transferrin receptor, in conjunction with IGSS techniques at the light microscope level, give results qualitatively similar to autoradiography, and the IGSS technique is simpler and faster than autoradiography.

At the electron microscope level, IGSS techniques are advantageous in that silver staining of gold labeled sections may be used to guide thin sectioning or thin sections may undergo direct postembedding silver enhancement. Finally, 1-nm labeled secondary antibodies, IGSS techniques, and bovine brain capillaries isolated with an enzymatic homogenization technique are combined to allow unique identification of brain capillary endothelial plasma membrane proteins at the ultrastructural level. (The J Histotechnol 16:249, 1993)     

New immunohistochemical "sandwich" staining method for mdr1 P-glycoprotein detection with JSB-1 monoclonal antibody in formalin-fixed,paraffin-embedded human tissues.

We have developed a new immunoperoxidase "sandwich" staining method for amplified detection of P-glycoprotein (Pgp) that is suitable for use on formalin-fixed, paraffin-embedded (conventional) tissue sections. This was accomplished by substantially changing the procedure described by Chan (1988) so as to increase specific staining intensity and to decrease nonspecific background staining. To determine the most appropriate primary antibody for the assay, we compared the immunoreactivity of JSB-1, C494, and C219 monoclonal antibodies recognizing internal epitopes of Pgp, and MRK16 and 4E3 monoclonal antibodies recognizing external epitopes of Pgp. Paraffin sections of Pgp-positive normal human tissues (adrenal, liver, kidney, and brain), of renal tumors, and of cell pellets of sensitive and multidrug resistant human tumor cell lines (MCF-7, KB) were used for comparisons. Immunostaining was excellent with JSB-1, moderate with C494, and very weak with C219. MRK16 and 4E3 showed no reaction. Nonspecific background staining was reduced by 1) omitting immunoglobulin G from secondary antibodies; 2) decreasing the concentration of peroxidase-antiperoxidase complex; and 3) utilizing casein solution for blocking and washing. Pretreatment of sections before immunostaining was also simplified. Using JSB-1, the threshold for detection of elevated Pgp corresponded to less than two-fold relative resistance to doxorubicin. Applying this method, we found two of 26 non-small cell lung cancers were positive for Pgp, consistent with previous results of others using frozen sections. This new immunoperoxidase sandwich staining method using JSB-1 now allows reliable Pgp detection in sections of formalin-fixed, paraffin-embedded (archived) surgical specimens and small biopsy materials commonly used for diagnostic purposes.