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.     

Ultrastructural localization of tubulin and actin in polyethylene glycol-embedded rat seminiferous epithelium by immunogold staining

The polyethylene glycol (PEG) method for immunofluorescence localization of cytoskeletal antigens has been extended to the ultrastructural level using glutaraldehyde-fixed tissues and immunogold staining. Semithin sections of fixed tissue embedded in polyethylene glycol are divested of the PEG, exposed to purified antibodies (e.g., antiactin, antitubulin) and anti-IgG-colloidal gold. The sections may be processed by dehydration and critical-point drying, or reembedment in hydrophilic substances. Tubulin is demonstrated in the mitotic spindles of dividing spermatogonia, manchettes, axonemes and centrioles of developing spermatids, and in the Sertoli cell cytoplasm; actin localization is demonstrated in the myoid cells of the tunica propria, and smooth muscle cells of arterioles in the interstitial tissue. The results demonstrate the applicability and versatility of PEG embedding for immunocytochemistry.     

Ultrastructural localization of tubulin and actin in polyethylene glycol-embedded rat seminiferous epithelium by immunogold staining

The polyethylene glycol (PEG) method for immunofluorescence localization of cytoskeletal antigens has been extended to the ultrastructural level using glutaraldehyde-fixed tissues and immunogold staining. Semithin sections of fixed tissue embedded in polyethylene glycol are divested of the PEG, exposed to purified antibodies (e.g., antiactin, antitubulin) and anti-IgG-colloidal gold. The sections may be processed by dehydration and critical-point drying, or reembedment in hydrophilic substances. Tubulin is demonstrated in the mitotic spindles of dividing spermatogonia, manchettes, axonemes and centrioles of developing spermatids, and in the Sertoli cell cytoplasm; actin localization is demonstrated in the myoid cells of the tunica propria, and smooth muscle cells of arterioles in the interstitial tissue. The results demonstrate the applicability and versatility of PEG embedding for immunocytochemistry.     

Lymphocyte membrane antigens in glycol methacrylate embedded tissue

The use of formalin or Michel's solution either alone or in combination with acetone, and acetone, methanol or ethanol alone as fixatives, and glycol methacrylate as embedding medium were evaluated for their suitability in procedures to detect lymphocyte membrane antigens by OKT and Leu monoclonal antibodies in human tonsils. No staining was detected in sections fixed in 70% or absolute ethanol and embedded in glycol methacrylate with either the direct immunofluorescence or avidin-biotin methods. Fixation in Michel's solutions plus acetone at room temperature revealed staining by both. Neither method resulted in staining after fixation in Michel's solution plus acetone at 4 C presumably due to the slow action of the fixative. Staining was enhanced using a combination of primary and secondary biotinylated antibodies. Dual staining allowed concurrent detection of two antigens in the same section. Glycol methacrylate embedding is a possible replacement for ultracold storage in the preservation of tissue for immunofluorescent staining.     

Lymphocyte Membrane Antigens in Glycol Methacrylate Embedded Tissue

The use of formalin or Michel's solution either alone or in combination with acetone, and acetone, methanol or ethanol alone as fixatives, and glycol methacrylate as embedding medium were evaluated for their suitability in procedures to detect lymphocyte membrane antigens by OKT and Leu monoclonal antibodies in human tonsils. No staining was detected in sections fixed in 70% or absolute ethanol and embedded in glycol methacrylate with either the direct immunofluorescence or avidin-biotin methods. Fixation in Michel's solutions plus acetone at room temperature revealed staining by both. Neither method resulted in staining after fixation in Michel's solution plus acetone at 4 C presumably due to the slow action of the fixative. Staining was enhanced using a combination of primary and secondary biotinylated antibodies. Dual staining allowed concurrent detection of two antigens in the same section. Glycol methacrylate embedding is a possible replacement for ultracold storage in the preservation of tissue for immunofluorescent staining.     

Gene transfer method for transient gene expression, stable transformation, and cotransformation of suspension cell cultures.

A new method was developed to study transient gene expression, stable transformation, and cotransformation in suspension cells, such as mouse myeloma and erythroleukemia cells. This method involves attachment of cells to a concanavalin A-coated tissue culture dish, treatment of cells with DEAE-dextran to adsorb plasmid DNA to the attached cells, and finally treatment with a 40% solution of polyethylene glycol to facilitate the uptake of DNA by the cells. Plasmids pSV2cat and pSV2neo were used as markers to optimize the conditions for transient gene expression and stable transformation, respectively, of mouse myeloma and erythroleukemia cells. This method was successfully used to obtain cotransformants of mouse myeloma cells.     

Extraction of Tissue Antigens for Functional Assays

Many of the antigen targets of adaptive immune response, recognized by B and T cells, have not been defined ¹. This is particularly true in autoimmune diseases and cancer². Our aim is to investigate the antigens recognized by human T cells in the autoimmune disease type 1 diabetes ^1,3,4,5. To analyze human T-cell responses against tissue where the antigens recognized by T cells are not identified we developed a method to extract protein antigens from human tissue in a format that is compatible with functional assays ⁶. Previously, T-cell responses to unpurified tissue extracts could not be measured because the extraction methods yield a lysate that contained detergents that were toxic to human peripheral blood mononuclear cells. Here we describe a protocol for extracting proteins from human tissues in a format that is not toxic to human T cells. The tissue is homogenized in a mixture of butan-1-ol, acetonitrile and water (BAW). The protein concentration in the tissue extract is measured and a known mass of protein is aliquoted into tubes. After extraction, the organic solvents are removed by lyophilization. Lyophilized tissue extracts can be stored until required. For use in assays of immune function, a suspension of immune cells, in appropriate culture media, can be added directly to the lyophilized extract. Cytokine production and proliferation by PBMC, in response to extracts prepared using this method, were readily measured. Hence, our method allows the rapid preparation of human tissue lysates that can be used as a source of antigens in the analysis of T-cell responses. We suggest that this method will facilitate the analysis of adaptive immune responses to tissues in transplantation, cancer and autoimmunity.     

Ultrastructural immunocytochemistry of glia cells. Double labeling studies using LR White embedding and colloidal gold

The introduction of acrylate resins (Lowicryl K4M, LR White) into electronmicroscopic immunocytochemistry applied to embedded tissue (post-embedding method) has improved the localization of antigens because of a satisfactory preservation of both ultrastructure and antigenicity of tissues. Here we describe a method that allows double staining of intracellular and membranous determinants in ultrathin sections of nervous tissue and cultures of peripheral nervous system cells. Ultrathin sections of the rat central nervous system fixed on uncoated grids were stained first for MBP selectively on the one face, then the opposite face was stained for GFAP using monoclonal antibodies and indirect immunogold staining method (IGS). Cultured Schwann cells induced to express major histocompatibility complex (MHC) class II antigens were stained for class II antigens by pre-embedding method then followed by post-embedding IGS for the other intracytoplasmic antigens.     

Immunohistochemical Double Staining of Microwave Enhanced and Nonenhanced Nuclear and Cytoplasmic Antigens

Many of the antigens commonly investigated in histopathology can be enhanced by microwave pretreatment (MWPT) of formalin fixed, paraffin embedded tissue sections. We developed a double labeling method using microwave heating to detect otherwise undetectable nuclear antigens combined.with immunohisto-chemistry (IHC) of cytoplasmic or membranous antigens that do not benefit from MWPT. We used the same primary antibody solutions used in single antibody IHC. The staining technique is based on the alkaline phosphatase anti-alkaline phosphatase (APAAP) and the labeled avidin-biotin (LSAB) methods. Four different protocols were tested, each modifying the sequence of MWPT, APAAP and LSAB staining. In this study Ki67, estrogen receptor, progesterone receptor, c-neu, CD68 and desmin primary antibodies were used in routinely formalin fixed, paraffin embedded tissues of 50 tumor specimens. MWPT followed by LSAB for microwave enhanced antigens and APAAP for antigens that cannot be enhanced by MWPT gave the best double staining results. This method improves characterization of tumor cell features from paraffin embedded tissue and should aid analysis of tumor differentiation, receptor status and nuclear proteins in the single cells in archival tissues.     

Resin embedment of organs and postembedment localization of antigens by immunoperoxidase methods

Various procedures for nonpolar and polar resin embedment were applied to mouse and rat livers for the study of postembedment immunolocalization of alpha 1-fetoprotein, albumin and the microsomal enzyme epoxide hydrolase. Fixations with formaldehyde and with formaldehyde-glutaraldehyde mixtures were used for tissue stabilization. Both fixation schedules did not abolish immunoreactivity. Treatment of liver with inert compounds such as polyvinylpyrrolidones or chemical modification of antigens with ethyl acetimidate prior to embedment improved immuno-staining. Either the low-polarity solvent ethanol or the highly polar ethylene glycol could be employed as dehydrating agents. Antigens were readily localized in sections from Epon 812 embedded livers. For this purpose, polymerized resin had to be partially removed. On the other hand, immunoreactivity of antigens was only faint after embedment in an epoxy-resin based on diepoxide octane. Also, antigens reacted faintly in sections from livers which were embedded at 0 degrees C in the polar acrylate-methacrylate based Lowicryl K4M resin. The indirect peroxidase labelled antibody method was as specific and sensitive as the PAP technique. Optimal antigen detection was attained with antibodies isolated by affinity chromatography and purified peroxidase conjugates. Apart from purified immunological reagents, the addition of high molarity sodium chloride and bovine serum albumin to the wash solutions enhanced immunohistological specificity.     

Ultrastructural immunocytochemistry of glia cells

Summary The introduction of acrylate resins (Lowicryl K 4M, LR White) into electronmicroscopic immunocytochemistry applied to embedded tissue (post-embedding method) has improved the localization of antigens because of a satisfactory preservation of both ultrastructure and antigenicity of tissues. Here we describe a method that allows double staining of intracellular and membranous determinants in ultrathin sections of nervous tissue and cultures of peripheral nervous system cells. Ultrathin sections of the rat central nervous system fixed on uncoated grids were stained first for MBP selectively on the one face, then the opposite face was stained for GFAP using monoclonal antibodies and indirect immunogold staining method (IGS). Cultured Schwann cells induced to express major histocompatibility complex (MHC) class II antigens were stained for class. H antigens by pre-embedding method then followed by post-embedding IGS for the other intracytopasmic antigens.The Clinical Research Unit for Multiple Sclerosis is supported by Hermann and Lilly Schilling foundation     

Freeze-drying of bone tissue: immunocytochemistry and enzyme histochemistry on paraffin embedded and low-temperature resin embedded specimens.

A simple protocol of tissue preparation was sought, which would enable marker enzymes of bone cells and extracellular matrix antigens to be localized in the same tissue section with high optical resolution. For this purpose, snap-frozen samples of rat fetal skeletal tissues were dried in a FDU 010 freeze-drying unit (Balzers) for 8-12 h at -50 to -40 degrees C and 0.02 bar. Freeze-dried tissues were either vacuum-infiltrated at 45 degrees C and embedded undemineralized in Paraplast, or vacuum-infiltrated overnight at 4 degrees C and embedded undemineralized in glycol methacrylate. These procedures enabled enzyme cytochemistry for alkaline phosphatase and tartrate-resistant acid phosphatase, and immunocytochemical staining for collagen types I, III, and laminin to be performed on the same sections. No pretreatment of the sections was necessary to reveal collagen antigenicity. This study reveals the possibility of complementing immunocytochemical studies of extracellular matrix with enzyme cytochemistry and, above all, with the excellent tissue preservation and high resolution afforded by plastic embedding.     

Ultrastructural localization of human kidney antigens using monoclonal antibodies.

A highly sensitive method of ultrastructural-immunoperoxidase staining was developed for use with monoclonal antibodies which have been raised in this laboratory to a variety of antigens of the human kidney. Because of the susceptibility of the antigens to fixation and processing, a four layer, pre-embedding method of staining was used. Results confirmed and clarified previously reported light microscopy results, indicating that an antigen recognized by the PHM5 antibody was found on the podocyte cell membrane within the glomerulus and was not present within the glomerular basement membrane. The antigen was also present on the extraglomerular endothelial cell membrane. The study also demonstrated the presence of an antigen specific to endothelial cells throughout the renal cortex, and gave further insight into the precise localization of glomerular basement membrane components including fibronectin. The method of staining is now being used together with detailed ultrastructural studies to identify the cells produced from isolated glomeruli in tissue culture.     

The identification of HL-A antigens on fresh and cultured human endothelial cells.

Human endothelial cells were obtained from the umbilical cord veins of 16 newborns by methods previously described and tested for HL-A antigens by a microcytotoxicity method. HL-A antigens were present on all endothelial cell lines tested. When the HL-A phenotypes of fresh endothelial cells and autologous fetal lymphocytes were compared, a concordance of 70% was observed. When the HL-A phenotypes of maternal lymphocytes and fresh endothelial cells were compared, a maternal contribution to the endothelial cell phenotype was evident in 72% of the possible commmon antigens. Some HL-A antigens were deleted from 11 of 16 endothelial cell lines that were re-typed after 2 weeks in tissue culture. The majority (90%) of deleted antigens were from the second HL-A locus. When three lines of endothelials cells were again re-typed after 6 weeks in culture, no further changes in antigenicity were noted. These findings: a) demonstrate that HL-A antigens are present on human endothelium and suggest that endothelial cells are actively involved in establishing the immunogenicity of a graft, and b) demonstrate that the HL-A antigens on human endothelial cells may be modulated by in vitro culture.     

A novel method for monitoring protoplast fusion

Summary Two fluorescent compounds, scopoletin and carboxyfluorescein, have been used to label both tissue culture and leaf mesophyll cells and protoplasts. The compounds localized within the vacuoles of cells in approximately 15 hours. They remained in the vacuole during cell wall digestion, and fluorescence was observable for several hours after protoplast release. A one day pulse of these fluorescent labels had no deleterious effect on the growth of cells or protoplasts. When morphologically indistinguishable protoplasts were labeled and treated with polyethylene glycol, multicolored fluorescent fusion products were observable. These fluorescent labels provide a convenient method for selection of heterokaryon fusion products of whole plant and tissue culture cell protoplasts.     

Characterization of fibroblast-like cells from the rat olfactory bulb

The isolation and characterization of stem cells from an alternative tissue is a subject of intensive investigation. In the present study, we have focused on the characterization of fibroblastic cells in olfactory bulb tissue of the rat. To this end, 4-6 week old rats were killed and their olfactory bulb tissue was dissected out. Olfactory bulb derived fibroblast-like cells were recovered by adhesion to cell culture plastic. The plastic adherent cultivated cells were induced to differentiate along osteoblastic, adipogenic and chondrogenic lineages. Furthermore, the expression of some surface antigens was investigated. We obtained purified cells with spindle shaped morphology in primary culture, which differentiated into mesenchymal lineages. These cells expressed CD29 and CD90 (Thy1.1) surface antigens, but not CD31, CD34 and CD45. Our results indicate that fibroblast-like cells from the olfactory bulb are mesenchymal stem cells in nature. Taken together, our data suggest that olfactory bulb tissue may constitute a new source of mesenchymal stem cells and could be used for the treatment of injury.     

Resin embedment of organs and postembedment localization of antigens by immunoperoxidase methods

Summary Various procedures for nonpolar and polar resin embedment were applied to mouse and rat livers for the study of postembedment immunolocalization of alpha₁-fetoprotein, albumin and the microsomal enzyme epoxide hydrolase. Fixations with formaldehyde and with formaldehyde-glutaraldehyde mixtures were used for tissue stabilization. Both fixation schedules did not abolish immunoreactivity. Treatment of liver with inert compounds such as polyvinylpyrrolidones or chemical modification of antigens with ethyl acetimidate prior to embedment improved immuno-staining. Either the low-polarity solvent ethanol or the highly polar ethylene glycol could be employed as dehydrating agents. Antigens were readily localized in sections from Epon 812 embedded livers. For this purpose, polymerized resin had to be partially removed. On the other hand, immunoreactivity of antigens was only faint after embedment in an epoxy resin based on diepoxide octane. Also, antigens reacted faintly in sections from livers which were embedded at 0° C in the polar acrylate-methacrylate based Lowicryl K4M resin. The indirect peroxidase labelled antibody method was as specific and sensitive as the PAP technique. Optimal antigen detection was attained with antibodies isolated by affinity chromatography and purified peroxidase conjugates. Apart from purified immunological reagents, the addition of high molarity sodium chloride and bovine serum albumin to the wash solutions enhanced immunohistological specificity.Supported by the Deutsche Forschungsgemeinschaft (Ku 257/3) Bonn, Federal Republic of Germany     

Light microscopic triple-colored immunohistochemical staining on the same vibratome section using the avidin-biotin-peroxidase complex technique

The sequential application of the avidin-biotin-peroxidase complex technique was used to localize multiple tissue antigens on a single free floating section of rat brain. Sequential visualization of individual antigens was achieved by the silver-gold-intensified diaminobenzidine (DAB) in the first step, nickel-intensified DAB in the second step, and the DAB alone in the third step of the immunostain procedure. For the demonstration of this method, tyrosine hydroxylase (TH), corticotropin-releasing factor (CRF), and vasopressin (VAS) antisera were used. Sections from the hypothalamic paraventricular nucleus (PVN) of rats pretreated with colchicine were stained. Black TH containing cell bodies were clearly distinguished from blue stained CRF cells and from yellow stained VAS-containing cell bodies in the PVN on the 25-30 micron thick vibratome sections. The sequential immunostaining procedure presented here results in superior staining of multiple antigens as compared to that achieved by the sequential application of the peroxidase-antiperoxidase (PAP) technique.     

Serological and genetic identification of a bovine B lymphocyte alloantigen system

A two-colour fluorescence micro cytotoxicity test was used to screen antisera for antibodies specific for bovine B lymphocytes. A total of 114 cattle alloantisera were screened against peripheral blood lymphocytes from 100 unrelated individuals. Anti-B lymphocyte activity was detected in 47 antisera. Cytotoxic antibodies to antigens other than B lymphocyte specific antigens were removed by absorbing the antisera with buffy coat cells or platelets isolated from whole blood. Selected antisera were used to type paternal half-sib families. The presence of a polymorphic, MHS-linked antigen system on B lymphocytes was demonstrated. The tissue distribution and MHS linkage of these antigens suggests this system is analogous to the class II or Ia antigens of other species.     

Serological and genetic identification of a bovine B lymphocyte alloantigen system

A two-colour fluorescence microcytotoxicity test was used to screen antisera for antibodies specific for bovine B lymphocytes. A total of 114 cattle alloantisera were screened against peripheral blood lymphocytes from 100 unrelated individuals. Anti-B lymphocyte activity was detected in 47 antisera. Cytotoxic antibodies to antigens other than B lymphocyte specific antigens were removed by absorbing the antisera with buffy coat cells or platelets isolated from whole blood. Selected antisera were used to type paternal half-sib families. The presence of a polymorphic, MHS-linked antigen system on B lymphocytes was demonstrated. The tissue distribution and MHS linkage of these antigens suggests this system is analogous to the class II or Ia antigens of other species.