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.     

Advances in ultrastructural postembedment localization of antigens in Epon sections with peroxidase labelled antibodies

Procedures for ultrastructural postembedment immunolocalization of antigens were investigated by use of the indirect peroxidase labelled antibody method. Results were compared with those obtained with the ultrastructural preembedment technique. IgG globulins in IgG synthesizing cells of rat lymph nodes served as model. Formaldehyde fixation, ethanol dehydration and embedment in Epon 812 did not abolish immunoreactivity. Sufficient numbers of antigens remained for subsequent postembedment immunohistology. Antigen binding sites were readily localized in ultrathin sections. For this purpose, polymerized resin had to be partially removed. Sodium methoxide in methanol/benzene mixture rapidly dissolved ultrathin sections. Diluted alcoholic sodium hydroxide enabled reliable resin etching and subsequent immunostaining. Treatment of ultrathin sections with hydrogen peroxide alone, did not permit immunolocalization of IgG. Optimal antigen detection was attained with antibodies isolated by affinity chromatography and purified peroxidase conjugates. IgG was stained in the rough-surfaced endoplasmic reticulum, the perinuclear space and the Golgi apparatus; the subcellular distribution corresponded to that obtained with preembedment immunohistology. In the latter technique, substrate accumulation was more homogenous than in postembedment staining.     

Advances in ultrastructural postembedment localization of antigens in Epon sections with peroxidase labelled antibodies

Summary Procedures for ultrastructural postembedment immunolocalization of antigens were investigated by use of the indirect peroxidase labelled antibody method. Results were compared with those obtained with the ultrastructural preembedment technique. IgG globulins in IgG synthesizing cells of rat lymph nodes served as model. Formaldehyde fixation, ethanol dehydration and embedment in Epon 812 did not abolish immunoreactivity. Sufficient numbers of antigens remained for subsequent postembedment immunohistology. Antigen binding sites were readily localized in ultrathin sections. For this purpose, polymerized resin had to be partially removed. Sodium methoxide in methanol/benzene mixture rapidly dissolved ultrathin sections. Diluted alcoholic sodium hydroxide enabled reliable resin etching and subsequent immunostaining. Treatment of ultrathin sections with hydrogen peroxide alone, did not permit immunolocalization of IgG. Optimal antigen detection was attained with antibodies isolated by affinity chromatography and purified peroxidase conjugates. IgG was stained in the rough-surfaced endoplasmic reticulum, the perinuclear space and the Golgi apparatus; the subcellular distribution corresponded to that obtained with preembedment immunohistology. In the latter technique, substrate accumulation was more homogenous than in postembedment staining.Supported by the Deutsche Forschungsgemeinschaft (Ku 257/5-2) Bonn, FRG     

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.     

Efficiency and sensitivity of indirect immunoperoxidase methods

The peroxidase-antiperoxidase (PAP) complex method has repeatedly been claimed to be more sensitive and antibody efficient than the indirect peroxidase labeled antibody method. However, most studies comparing these methods used tissue sections as the test material. However, test systems with known amounts of antigen will allow more reliable comparison of these methods and quantitative evaluation of method sensitivity. We therefore compared the antibody efficiency and sensitivity of these methods for the detection of human chorionic gonadotropin in an enzyme linked immunosorbent assay (ELISA), an antigen spot test (AST) and tissue sections of choriocarcinoma. In the PAP technique rabbit PAP and goat anti-rabbit antibody were applied. The same antibody was peroxidase-labeled with the periodate technique and used in the labeled antibody method. In the ELISA the PAP method resulted in slightly higher antibody efficiency than the labeled antibody method. At low primary antibody dilutions the intensity of the reaction decreased with the PAP method but remained high with the labeled antibody method, in the ELISA as well as on tissue sections. In the AST the labeled antibody method and the PAP method appeared to be equally sensitive.     

Subcellular location of the major protein antigens of paramyxoviruses revealed by immunoperoxidase cytochemistry

The major structural proteins of Newcastle disease virus and Sendai virus were localized in infected BHK-21 and MDBK cells by ultrastructural immunoperoxidase cytochemistry using antibodies against the individual viral protein antigens. The intracellular glycoproteins were strictly membrane bound, being localized in the rough endoplasmic reticulum (RER), perinuclear spaces, smooth membrane vesicles, and presumed Golgi apparatus. The nucleocapsid proteins were detected exclusively in membrane free cytosol and accumulated there, forming inclusions. The membrane (M) protein was found both in cytosol and on RER. The viral proteins on RER exhibited a distinct site specificity; the glycoproteins were facing the lumen of RER whereas M protein was present at the outer cytoplasmic surface. All the viral proteins were detectable at the plasma membrane where virus assembly takes place. However, their modes of distribution differed remarkably. The glycoproteins were spread widely over the entire cell surface including the areas of virus budding and those of normal morphology, whereas M protein was localized in restricted areas of the membrane, frequently forming a patch of virus specific membrane. The presence of nucleocapsids was confined to the virus particles budding from the plasma membrane. These results complement and extend the earlier morphological and biochemical data on the assembly or morphogenesis of paramyxoviruses.     

Ultrastructural localization of glial fibrillary acidic protein in mouse cerebellum by immunoperoxidase labeling

Glial fibrillary acidic protein was localized at the electron microscope level in the cerebellum of adult mice by indirect immunoperoxidase histology. In confirmation of previous studies at the light microscope level, the antigen was detectable in astrocytes and their processes, but not in neurons or their processes, or in oligodendroglia. Astrocytic processes were stained in white matter, in the granular layet surrounding synaptic glomerular complexes, and in the molecular layer in the form of radially oriented fibers and of sheaths surrounding Purkinje cell dendrites. Astrocytic endfeet impinging on meninges and perivascular membranes were also antigen positive. In astrocytic perikarya and processes, the immunohistochemical reaction product appears both as a diffuse cytoplasmic label and as elongated strands, which by their distribution and frequency could be considered glial filaments.     

Ultrastructural localization of guinea pig spermatozoal autoantigens on germinal cells by immunoperoxidase techniques.

Three guinea pig spermatozoal autoantigens S, P and T, each one able to induce autoimmune aspermatogenic orchiepididymitis and autoantibodies, were ultrastructurally localized in male germinal cells by immunoperoxidase techniques. Both living and prefixed sectioned cell preparations were treated and examined. Fab antibody fragments were used to study intracellular antigens (whole antibodies were inefficient). Water-soluble S and P autoantigens were found in acrosomal structures in the same sites: proacrosomal and acrosomal granules of the young spermatids, on the head caps of spermatids and acrosomal cap of spermatozoa, along the inner and outer acrosomal membranes and in the outer zone of the acrosomal matrix of the same cells. S was never found in the inner zone of spermatid or spermatozoa acrosomes, while P was present in this inner zone, but only of young spermatids. Water-insoluble T autoantigen was found on the plasmalemma and outer acrosomal membranes of spermatids and spermatozoa, inside the spermatid cytoplasm and, sometimes, on the inner acrosomal membrane of young spermatids. The specificity of the immunological localization for each antigen was confirmed by testing with specific antisera following absorption with homologous and heterologous antigens. No other testicular cell type (including Sertoli cells per se) was found to bear S, P or T autoantigens. When use was made of autoimmune sera obtained through autologous whole spermatozoa, the observed staining was an additive combination of what was observed when using the preceding three immune sera, anti-S, anti-P and anti-T.     

Microphotometric quantitation of the reaction product of several indirect immunoperoxidase methods demonstrating monoclonal antibody binding to antigens immobilized on nitrocellulose

The nitrocellulose model and microphotometry were used to investigate whether in immunoperoxidase cytochemical methods the amount of final reaction product reflects the amount of cell surface antigen. The results obtained with four cytochemical peroxidase methods, i.e., those using diaminobenzidine/H2O2 (DAB/H2O)2, DAB/H2O2/COCl2, DAB/H2O2/imidazole, and silver intensification of the DAB end product, were compared first. The quantitative DAB/H2O2/imidazole method proved to be the most sensitive and was selected for further studies. Cell surface antigens prepared by solubilization of peritoneal macrophages with octyl-beta-D-glucopyranoside were immobilized on nitrocellulose. Monoclonal antibody binding to these cell antigens was detected by peroxidase immunocytochemistry. Comparison of the sensitivity of the indirect immunoperoxidase and the biotin-(strept)avidin immunoperoxidase methods on the basis of the highest detectable dilution of a cell lysate showed that these methods were equally sensitive. A linear relationship between the absorbance of the peroxidase reaction product and the amount of cell lysate immobilized on nitrocellulose was found for all three indirect immunoperoxidase methods. This proves that the amount of final immunocytochemical peroxidase reaction product is proportional to the amount of antigen in cell lysates. However, the relative expression of antigens in intact cells differs from that in cell lysates. Therefore, the present method to solubilize cells and immobilize cell antigens cannot be used to quantitate the antigen content of cells.     

Postembedding immunocytochemical localization of paramyxovirus antigens by light and electron microscopy

A postembedding method is described to localize antigens specific for various paramyxoviruses in sections of cells and tissues that have been fixed and embedded in epoxy resins for conventional electron microscopy. Viral antigens were localized in CV-1 cell cultures infected with simian virus 5 (SV5), brains of suckling hamsters inoculated with either neuroadapted mumps virus or hamster-adapted measles virus, and brains of adult mice infected with Sendai (parainfluenza I) virus. Both 1-micrometer-thick and thin (gold) tissue sections were etched with alcoholic sodium hydroxide-solution and then treated following either the unlabeled antibody peroxidase-antiperoxidase or the biotinylated protein A:avidin peroxidase procedure. Primary reagents included immunoglobulin isolated from hyperimmune rabbit sera with specificity to the major viral components of SV5 or SV5 hemagglutinin-neuraminidase, to whole mumps virus or mumps virus nucleocapsids, and to whole Sendai virus. Crude rabbit anti-Sendai virus antiserum and whole human subacute sclerosing panencephalitis (SSPE) sera were used in parallel. The results indicate that tissues processed for conventional evaluation by electron microscopy may be suitable, within limits, for postembedding immunocytochemical staining of paramyxovirus antigens.     

Simultaneous visualization of two antigens in the same tissue section by combining immunoperoxidase with immunofluorescence techniques.

A simple method was developed whereby immunoperoxidase and immunofluorescence techniques were applied in consecutive steps to demonstrate the presence of two antigens in the same tissue section. This method was applied in three model, two antigens were shown: a) each (gastrin and pepsinogen II) inside one of two different cell types (gastrin (G) and antral peptic cells), b) each (kappa or gamma light chains) inside different cells of the same type (plasma cells); also, both (kamma and gamma light chains) inside the same cell (Reed-Sternberg cell), and c) both (pepsinogen I and II) inside the same cell (chief cell of oxyntic glands). The results could be viewed and photographed either simultaneously, when the antigens were in different cells, or sequentially, when the antigens were in the same cells.     

Heterogenetic antigens of Yersinia pseudotuberculosis in human erythrocytes and organs

The study of the antigenic relationship between Y. pseudotuberculosis in red blood cells and different highly vulnerable tissues of the human body (in the liver, the spleen, the kidneys, the lymph nodes, the intestine) has been carried out; as a result, the presence of heterogeneous substances has been shown. Y. pseudotuberculosis heterogeneous antigens play an important role in the formation of severe forms of the disease and determine the frequency of relapses; they also determine the specific action of diagnostic and therapeutic preparations.     

Improvements in immunostaining samples embedded in methacrylate: localization of microtubules and other antigens throughout developing organs in plants of diverse taxa

Microtubules are important in plant growth and development. Localizing microtubules in sectioned material is advantageous because it allows any tissue of interest to be studied and it permits the positional relations of the cells within the organ to be known. We describe here a method that uses semi-thin (0.5–2 m) sections of material embedded in butyl-methylmethacrylate, to which 10 mM dithiothreitol was added. After removing the embedding material and using indirect immunofluorescence staining, we obtain clear images of microtubules, actin microfilaments, callose and pulse-fed bromodeoxyuridine. This method works on the root tissues of Arabidopsis thaliana(L.) Heynh, Pinus radiataD. Don, Zamia furfuraceaAit., Azolla pinnataR. Br. and on sporophytic tissues of Funaria hygrometricaHedw. In general, most of the cells in the organs studied are successfully stained. Using this method, we find that interphase meristematic cells in all of these species have microtubules not only in the usual cortical array but also throughout their cytoplasm. The presence of the calcium chelator ethylene glycol-bis(-aminoethyl ether)N,N,N,N-tetraacetic acid EGTA in fixation buffers led to some tissue damage, and did not enhance the preservation of microtubules. The common assumption that EGTA-containing buffers stabilize plant microtubules during fixation appears unwarranted.Abbreviations BrdU 5-bromodeoxyuridine - DTT dithiothreitol - EGTA ethylene glycol-bis(-aminoethyl ether) - N,N,N,N tetraacetic acid We thank Ann Cork for technical assistance, Professor B.E.S. Gunning (Australian National University) and Drs. A.R. Hardham (A.N.U.) and R.E. Williamson (A.N.U.) for intellectual and material support, Dr D. McCurdy (A.N.U.) for the purified anti-actin antibody, and Professor B. Stone (La Trobe University, Melbourne, Australia) for generously providing the anti-callose antibody. We also thank the Electron Microscopy Unit of A.N.U. for the use of facilities. L.C.F. gratefully acknowledges financial support from the National Sciences and Engineering Research Council of Canada.     

Ultrastructural localization of gonadotrophic hormones in the porcine pituitary using the immunoperoxidase technique

Summary Using specific antibodies against subunits of porcine LH and FSH, the pituitary cells which produce these two gonadotrophins were identified in the porcine pituitary at the ultrastructural level using the immunoperoxidase technique. It was clearly shown that most of the gonadotrophic cells are responsible for the production of both FSH and LH. However, some cells, only positive for FSH or LH, indicate that the concentrations of FSH and LH vary from cell to cell. At the ultrastructural level, the FSH/LH cells contain one class of secretory granules strongly positive for both FSH and LH as well as large negatively stained dense bodies. These findings indicate that the one cell-one hormone concept may not apply to gonadotrophic hormones; a FSH/LH cell cannot be distinguished from a LH or a FSH cell without immunocytochemical identification of its hormonal content.Abbreviations p-LH porcine luteinizing hormone - p-LH porcine LH subunit - p-LH porcine LH subunit - p-FSH porcine follicule stimulating hormone - p-FSH porcine FSH subunit - b-TSH bovine thyrotropic hormone