Tissue preparation for the demonstration of surface antigens by immunoperoxidase techniques

Summary The fixation and drying regimes for frozen sections and cytocentrifuge preparations for the demonstration of surface antigens, such as immunoglobulins and iron binding proteins, vary enormously between different groups of workers. A method using freeze-dried sections and acetone fixation was compared with 16 other methods of fixation and found to be the best for tissue preservation and antigen demonstration. Freeze drying was found to improve the cytological preservation of air-dried sections considerably.     

A new histological embedding method by low-temperature polymerisation of methyl methacrylate allowing immuno- and enzyme histochemical studies on semi-thin sections of undecalcified bone marrow biopsies

Summary A new procedure of embedding in methyl methacrylate (MMA) is introduced, which enables immunostaining by preservation of cellular epitopes. This could be achieved by reduction of polymerisation temperature from ca. 60° C to 22° C within the core of tissue blocks. Reduction of the polymerisation temperature is due to destabilisation of acrylate monomer, reduction of catalyst, exclusion of molecular oxygen, chemical initiation and reduction of environmental temperature. This results in good preservation of antigens and enzymes in the haematopoietic and lymphatic tissue of bone marrow as well as lymphoid, epithelial and mesenchymal markers in other tissues, comparable to paraffin embedding. Results are demonstrated by application of monoclonal and polyclonal antibodies and by demonstration of enzyme activity conventionally used in haematology.     

Fixation, processing, and immunochemical reagent effects on preservation of T-lymphocyte surface membrane antigens in paraffin-embedded tissue

Fixatives, fixation additives, paraffin processing reagents, and immunochemical reagents were investigated for effects on preservation of T-lymphocyte surface membrane antigens CD3, CD4, and CD8 in human tonsil. Individual reagent effects were assessed in frozen sections by use of monoclonal antibodies and this information was used to optimize T-cell immunostaining in paraffin sections. Harmful factors were fixation delay, fixation at acid pH, fixation and processing at temperatures above 4 degrees C, hot paraffin wax, proteolytic enzymes, methanolic hydrogen peroxide, Triton X-100, and prolonged iodine treatment. Optimal T-cell demonstration in paraffin sections followed tissue fixation in periodate-lysine-paraformaldehyde dichromate at 4 degrees C, pH 7.5; processing through isopropanol, then xylene or chloroform, at 4 degrees C; and embedding in low melting point wax at 45-50 degrees C. Graded antigen stability occurred: CD3 most stable, CD8 least, and CD4 intermediate. CD4 and CD8 antigen preservation in paraffin sections required critical optimal tissue handling. CD3 was more stable and was also demonstrated in tissue fixed in commercial formalin, glutaraldehyde, and Bouin's fluid when fixation and processing conditions were optimized for pH and temperature. Of the fixation additives studied, polyethylene glycol and several potassium and magnesium salts enhanced immunostaining, whereas calcium chloride and lidocaine were deleterious.     

Identification of leucocyte surface antigens in paraffin- embedded bovine tissues using a modified formalin dichromate fixation

A modified fixative of formalin dichromate was combined with a cold embedding procedure for the preservation of bovine leucocyte surface antigens. Fourteen monoclonal antibodies recognizing seven bovine leucocyte surface antigens (BoCD1w2, BoCD4, BoCD8, BoWC1, BoWC3, BoWC4 and BoIgM) were applied as primary antisera in a sensitive avidin--biotin--peroxidase complex detection method. The staining results were compared with those obtained in cryostat and routinely formalin-fixed sections of corresponding tissue samples. Using the modified formalin dichromate fixative and the cold embedding procedure, all the leucocyte surface antigens tested were detectable immunohistologically in paraffin sections with a generally more distinct staining than in traditionally processed tissues. Morphological structures were better preserved than in cryostat sections but, to some extent, were poorer when compared with routinely formalin-fixed tissues. However, this method suggests that there are only mild masking effects and provides an alternative to the use of unfixed material, particularly for morphological--immunohistochemical investigations     

Immunoelectron microscopic demonstration of tissue antigens with monoclonal antibodies

Two methods of demonstrating tissue antigens by ultrastructural enzyme immunohistochemistry were tested. The monoclonal antibodies Ki-M1 and Ki-M4 were chosen for testing the methods because Ki-M1 identifies a relatively stable, and Ki-M4 a very unstable antigen. The two antibodies react selectively with human macrophages and interdigitating reticulum cells or dendritic reticulum cells of lymphoid follicles. The Ki-M1 reaction product is confined to the surface membrane. Ki-M4 reactivity is located on the surface membrane and, less often and to a lesser extent, in the cytoplasm. The technical prerequisites for reliable conservation of the antigens identified by these two antibodies were standardized. The results indicated that prior fixation in 4% paraformaldehyde is preferable for optimum preservation of stable antigens. Application of the primary antibody prior to fixation was found to be the best procedure for demonstrating unstable antigens, although nonspecific reactions were seen more often with this method.     

Application of Microwave Irradiation to Immunohistochemistry: Preservation of Antigens of the Extracellular Matrix

Microwave irradiation as a means of fixation was evaluated for the preservation of extracellular matrix antigens such as collagen III, IV, fibronectin and laminin in both lung and liver specimens. Small tissue samples were placed in normal saline or periodatelysine-paraformaldehyde (PLP) and irradiated for 30 sec to bring them to a temperature of 50 C. The tissue was then processed rapidly in a tissue processor adjusted to a 2 hr cycle and embedded in paraffin. Sections were immunostained. For comparison, routine cryostat sections as well as sections of formalin fixed tissue were used. Microwave irradiation in saline gave excellent morphological detail, comparable to that in formalin fixed tissue. All four antigens evaluated were well preserved without the necessity of prior pepsin digestion. Microwave fixation is promising for preservation of antigenicity and morphological detail, and considerably reduces the time required for processing.     

Application of microwave irradiation to immunohistochemistry: preservation of antigens of the extracellular matrix

Microwave irradiation as a means of fixation was evaluated for the preservation of extracellular matrix antigens such as collagen III, IV, fibronectin and laminin in both lung and liver specimens. Small tissue samples were placed in normal saline or periodate-lysine-paraformaldehyde (PLP) and irradiated for 30 sec to bring them to a temperature of 50 C. The tissue was then processed rapidly in a tissue processor adjusted to a 2 hr cycle and embedded in paraffin. Sections were immunostained. For comparison, routine cryostat sections as well as sections of formalin fixed tissue were used. Microwave irradiation in saline gave excellent morphological detail, comparable to that in formalin fixed tissue. All four antigens evaluated were well preserved without the necessity of prior pepsin digestion. Microwave fixation is promising for preservation of antigenicity and morphological detail, and considerably reduces the time required for processing.     

Colloidal gold immunostaining on deplasticized ultra-thin sections

We localized tissue antigens on ultra-thin sections by deplasticizing the sections while on the grid, incubating in primary antiserum followed by immunoglobulin-conjugated colloidal gold, and ultimately re-embedding in dilute Epon. This procedure permitted ultrastructural localization of tissue antigens that were previously masked by the embedding plastic surrounding tissue components. In addition, replacement of the plastic matrix on the thin section after immunostaining prevented development of the drying artifacts that occur in unsupported tissue sections. Optimal preservation of components in the tissue sections was achieved despite extensive steps involved in plastic removal and immunostaining. This method may be useful in situations where the number of exposed epitopes on the surface of a thin section is low. The procedure also allows the use of antisera at greater dilutions and provides enhanced immunostaining specificity with low background.     

A new histological embedding method by low-temperature polymerisation of methyl methacrylate allowing immuno- and enzyme histochemical studies on semi-thin sections of undecalcified bone marrow biopsies.

A new procedure of embedding in methyl methacrylate (MMA) is introduced, which enables immunostaining by preservation of cellular epitopes. This could be achieved by reduction of polymerisation temperature from ca. 60 degrees C to 22 degrees C within the core of tissue blocks. Reduction of the polymerisation temperature is due to destabilisation of acrylate monomer, reduction of catalyst, exclusion of molecular oxygen, chemical initiation and reduction of environmental temperature. This results in good preservation of antigens and enzymes in the haematopoietic and lymphatic tissue of bone marrow as well as lymphoid, epithelial and mesenchymal markers in other tissues, comparable to paraffin embedding. Results are demonstrated by application of monoclonal and polyclonal antibodies and by demonstration of enzyme activity conventionally used in haematology.     

Ia antigens in plastic-embedded tissues: a post-embedding immunohistochemical study

The aim of the present study was to establish a plastic embedding technique that makes possible the immunohistochemical demonstration of class II major histocompatibility complex (MHC) antigens (Ia antigens) in undecalcified joint tissues. Therefore a series of fixatives and dehydrating agents was tested for saving Ia immunoreactivity by post-embedding immunostaining of thin sections (2 microns) of rat tissues that had been embedded in glycol methacrylate (GMA), and by comparing with cryostat sections. An indirect immunoperoxidase and the avidin-biotin complex (ABC) technique were used. Combined with fixation by 4% formaldehyde, dehydration with GMA was found to give the best preservation of Ia antigenicity, followed by dehydration with ethylene glycol. The thinness of tissue sections facilitated the association of Ia antigens with different subcellular compartments in distinct cell populations. These various patterns are described.     

DNA preservation in Precolumbian remains from the American Southwest

Naturally desiccated precolumbian remains from the U.S. Southwest were examined for DNA preservation in a variety of tissue types. Histological examination was used to determine if condition of morphological preservation indicated a tissue choice for nucleic acid extraction. It was found that nucleic acids are regularly present in tissue from these samples and neither macroscopic nor microscopic tissue appearance is an indicator of DNA preservation. Extracted DNA from Southwest specimens was small (<500 bp) but demonstrably human. Future analyses of ancient DNA with allele specific oligonucleotide probes should provide answers to specific anthropological questions about Southwestern Amerindian population dynamics, evolution and connections to modern Pueblo peoples.     

The application of lipid-soluble stains in plastic-embedded sections

Summary The present study was designed to develop a toutine method for direct demonstration and precize localisation of lipid substances in tissue sections. A panel of lipid-rich tissues was fixed in 4% buffered formaldehyde, infiltrated, and embedded in the water-soluble plastics Technovit 7100, EFL-67, and JB-4. The use of alcohol containing fluids was avoided. Staining with the lipid-soluble dyes Sudan Black B and Oil Red O revealed excellent preservation of tissue lipids in Technovit 7100 embedded sections when compared with cryostat sections of the same tissue specimens. Lipid preservation in EFL-67 and JB-4 embedded sections was inconsistent, even when infiltration and polymerization procedures were performed at 4°C. Combination of lipid-soluble dyes with the periodic acid Schiff Jones' methenamine silver, or Gomori' reticulin method allowed for an exact localization of lipids in high-quality Technovit 7100 embedded sections. The procedure herein is casily applicable in routine histopathology practice.     

Ultrafast microwave energy fixation for electron microscopy

We demonstrate that microwave (MW) energy can be used in conjunction with chemical cross-linking agents to fix tissue blocks rapidly for electron microscopy in as brief a time as 26 msec. The optimal ultrafast MW fixation methodology involved immersing tissue blocks up to 2 mm3 in dilute aldehyde fixative and immediately irradiating the specimens in a 7.3 kW MW oven for 26-90 msec, reaching a fixation temperature range of 32-42 degrees C. Ultrastructural preservation of samples irradiated by MW energy was comparable to that of the control samples immersed in aldehyde fixative for 2 hr at 25 degrees C. Potential applications for this new fixation technology include investigation of rapid intracellular processes (e.g., vesicular transport) and preservation of proteins that are difficult to demonstrate with routine fixation methods (e.g., antigens and enzymes).     

The application of lipid-soluble stains in plastic-embedded sections

The present study was designed to develop a routine method for direct demonstration and precise localization of lipid substances in tissue sections. A panel of lipid-rich tissues was fixed in 4% buffered formaldehyde, infiltrated, and embedded in the water-soluble plastics Technovit 7100, EFL-67, and JB-4. The use of alcohol containing fluids was avoided. Staining with the lipid-soluble dyes Sudan Black B and Oil Red O revealed excellent preservation of tissue lipids in Technovit 7100 embedded sections when compared with cryostat sections of the same tissue specimens. Lipid preservation in EFL-67 and JB-4 embedded sections was inconsistent, even when infiltration and polymerization procedures were performed at 4 degrees C. Combination of lipid-soluble dyes with the periodic acid Schiff, Jones' methenamine silver, or Gomori' reticulin method allowed for an exact localization of lipids in high-quality Technovit 7100 embedded sections. The procedure herein is easily applicable in routine histopathology practice.     

Simultaneous and successive localization of two antigens in the same tissue section using 3,3'-diaminobenzidine and 1-naphthol basic dye

We describe two procedures for the simultaneous and successive localization of two antigens in the same tissue section. In the simultaneous staining procedure, the first antigen was localized using 3,3'-diaminobenzidine (DAB), while the second antigen was stained using the 1-naphthol basic dye (1-NBD) method. The colour of the second antigen depended on the basic dye used, and no mixing of colours was observed when the two antigens were localized in different cells or structures. However, sequential double staining proved to be more convenient for the demonstration of two antigens in the same cell. In this procedure, the first antigen was stained using 1-NBD, and the interesting microscopic fields were photographed. The basic dye was then completely removed, and the second antigen was stained using DAB.     

Influence of fixation and decalcification on the immunohistochemical staining of cell-specific markers in paraffin-embedded human bone biopsies

A number of fixation and decalcification procedures were evaluated to determine their suitability for immunohistochemistry on trephine samples of bone marrow after paraffin embedding. In particular, the immunoreactivity of antigens characteristic for various hematopoietic cell lines (immunoglobulin heavy and light chains for plasmacytoid cells; elastase for neutrophil myeloid cells; lysozyme, alpha-1-antitrypsin and alpha-1-antichymotrypsin for hystiocytic cells; leukocyte common antigen for lymphocytes; hemoglobin and glycophorin A for erythroid cells; Factor VIII-related antigen for thrombocytoid cells) as well as some antigens specific for epithelial tumors (CEA, 115D8, and keratin) were investigated. Fixation in a mercuric chloride-formaldehyde mixture followed by decalcification in acetic acid-formaldehyde-saline proved to be the best procedure for antigen preservation and retention of morphologic detail. Moreover, there is no need of trypsinization when using this procedure. The only exception was Factor VIII-related antigen in megakaryocytes, which was best demonstrated in trypsin-digested sections of formalin-fixed and acetic acid-decalcified biopsies.     

Immunohistochemical application of monoclonal antibodies against myelin basic protein and neurofilament triple protein subunits: advantages over antisera and technical limitations

Four monoclonal antibodies against guinea pig myelin basic protein (MBP), and four against subunits of bovine neurofilament triplet proteins (NF) were produced and their activity determined by enzyme-linked immunosorbant assay. The specificity and cross-reactivity of these eight monoclonal antibodies and one heterologous antiserum against each of the two central nervous system (CNS) antigens were examined in a histological study using the immunoperoxidase, antibody sandwich technique in rat and human brain tissue. Tissue sections were prepared from paraffin-embedded or fresh brain tissue that had been fixed with one of five different fixatives. The resulting immunoperoxidase labeling was then graded for intensity and examined for artifacts. One monoclonal antibody against MBP and one against NF resulted in labeling that was superior to that given by each of the antisera against their respective antigens. Of the five fixatives tested, a mercuric chloride-formalin solution gave the best preservation of these two antigens in rat and human brain tissue. The mercuric chloride-formalin solution was found to be superior to the other fixatives when immersion fixation was used, and was especially optimal when brains were perfused fixed. Three artifacts were encountered among the various antibody-fixative combinations that produced erroneous, but seemingly specific staining of Purkinje cells, neurons and axons, or astrocytes.     

Ultrastructural localization of intracellular antigens by the use of protein A-gold complex.

An immunocytochemical technique for the demonstration of intracellular antigens (secretory proteins) on thin sections is reported. Staphylococcal protein A which reacts with the Fc fragment of IgG molecules was labeled with colloidal gold as a marker. The antigenic sites were visualized on aldehyde-fixed and Epon-embedded tissue in a two step procedure. The specific antisera were applied to thin sections for binding to the antigens and then visualized by the protein A-gold complex. By using this technique different secretory proteins of the exocrine and endocrine pancreas were localized. The protein A-gold technique is proposed as a general method for visualization of antigenic sites on thin sections.     

Use of LR white resin for post-embedding immunolabelling of brain tissue.

The object of this study was to investigate the applicability of the acrylic resin 'LR White' to immunolabelling of various antigenic determinants in aldehydefixed rat CNS tissue. Antibodies were used, which worked well in paraffin sections and therefore were suitable to detect antigens resistant to complete dehydration and heat. Different LR White embedding protocols were employed in order to select the preparation conditions that adequately preserved both the antigenicity and fine structure. Specimens were completely dehydrated with up to 100% ethanol, which was followed by various infiltration times with LR White monomer. Polymerization of the resin was induced by heat, a chemical catalytic procedure (accelerator), or ultraviolet (UV) light. Paraffin, as well as semithin and ultrathin LR White sections were incubated with antibodies reacting to antigens located on the cell surface (stage-specific embryonic antigen-1; SSEA-1), within the plasma membrane (myelin basic protein), in the cytosol (HNK-1, S100 protein), in the cytoskeleton (GFAP, vimentin, neurofilament protein, INT-FIL), and in the extracellular matrix (laminin). All of the examined antigens were immunocytochemically detectable in paraffin-embedded material, while the carbohydrate moieties, HNK-1 and SSEA-1, were not immunoreactive in LR White sections. However, in cryostat sections processed for pre-embedding immunoelectron microscopy, the HNK-1 epitope and SSEA-1 were immunolabelled. Polymerization carried out under UV light led to better structural preservation of brain tissue than resin cured with heat or catalyst. The length of prior infiltration with monomer apparently had no effect on tissue preservation. Consequently, UV light-induced polymerization of LR White gives acceptable morphology of brain tissue. However, the use of this acrylic resin is restricted to the detection of some CNS antigens only.     

Ultrastructural localization of HTLV-I gag proteins p19 and p24 by single and double immunogold labeling

We developed a post-embedding immunogold labeling procedure for the ultrastructural localization of the HTLV-I gag proteins p19 and p24 by the use of monoclonal antibodies (MAb). Both antigens were shown to withstand fixation with 1% glutaraldehyde. In addition, p19 antigenicity was found not to be affected by post-fixation with 1% osmium tetroxide. The choice of resin played a decisive role in the retention of antigenicity. P19 was preserved in Lowicryl K4M as well as in LR White, whereas p24 was preserved only in Lowicryl. Both p19 and p24 were found to be localized on the HTLV-I virions themselves, whereas no positive immunostaining could be observed on the infected cells. In Lowicryl-embedded samples, in which both antigens had been preserved, a double immunogold labeling procedure was performed that allowed the co-localization of p19 and p24 on the same section. In osmicated LR White-embedded samples the quality of ultrastructural preservation of HTLV-I virions was found to be comparable to results obtained with the traditional glutaraldehyde-osmium tetroxide-epoxy resin processing.