Localisation of enteropathogens in paraffin embedded tissue by immunoperoxidase

An indirect immunoperoxidase technique has been used to identify enteropathogens in formol-sublimate fixed paraffin embedded sections of calf intestine. Infections with bovine rotavirus, bovine coronavirus, Newbury agent SRV -1, and K99+ Escherichia coli have been detected in the intestines from experimentally infected and conventially reared diarrhoeic or normal calves. The ability to visualize enteropathogenic agents in histological sections resulted in the demonstration of virus infected cells at sites not previously shown to be infected using the immunofluorescence technique.     

Subcellular localization of HMFG2 in breast carcinomas: an immunohistochemical and immunoelectron microscopic study.

Malignant transformation of human cells is associated with morphological and biochemical alterations. We have studied the distribution and pattern of staining of HMFG2 (human milk fat globulin) in normal breast, benign breast lesions, and 137 primary and metastatic breast carcinomas. Immunohistochemical staining was performed with an antibody to HMFG2 using the indirect peroxidase technique. Three patterns of staining were noted: 1) secretion and luminal staining (in normal breast, most benign breast lesions and some breast carcinomas); 2) plasma membrane staining (in breast carcinomas); 3) intracytoplasmic staining (in breast carcinomas). Immunoelectron microscopy was also performed on normal breast, infiltrating duct, and lobular carcinomas. Immunoelectron microscopy showed localization of the gold particles on the electron dense granules of the HMFG2 protein. These were localized along the surface of the extracytoplasmic lumina in normal breast ducts/acini and breast carcinomas, whereas localization was also noted within the intracytoplasmic lumina in cancer cells only. These results show that there is altered localization of milk fat globulin in breast cancer cells associated with membrane internalization and formation of intracytoplasmic lumina. This contributes to the understanding of the phenotypic alterations associated with malignant transformation in breast cancer.     

The use of post-embedding immunoelectron microscopy in the diagnosis of glomerular diseases. Comparison of immunoelectron microscopic and immunofluorescence studies.

Fifty renal biopsies were studied by immunoelectron microscopy after embedding in a partly hydrophilic polyacrylic resin (LR White). Immunofluorescence studies were carried out on frozen sections of parallel tissue samples. Polyacrylic embedding gave good preservation of the renal ultrastructure and precise localization of immunoglobulin and C3c antibodies within glomerular electron-dense deposits. Non-specific staining of plasma proteins within vascular lumina could easily be detected. There was good correlation between immunoelectron and immunofluorescence microscopy. Immunoelectron microscopy is a very sensitive method, which can detect small amounts of antigen. More cases were, however, positive by immunofluorescence than by immunoelectron microscopy. This discrepancy may be explained by difference in sample size, and by difference in resolution of morphological details (electron microscopy versus fluorescence microscopy).     

Characterization of tissue amyloid by immunofluorescence microscopy

Immunohistochemical classification of amyloid type was possible in 44 of 50 (88%) patients as judged by the concordance of immunofluorescence, clinical, serum, and urine immunoelectrophoresis, and bone marrow data. In frozen tissue sections incubated with a panel of antisera monospecific for immunoglobulin heavy chains, kappa and lambda light chains, and amyloid-A-related protein, the amyloid was classified as AL in 20 and AA in 24. In 6 patients the amyloid could not be classified because of the absence of reactivity in 2 and overlap staining in 4. The findings indicate that routine immunofluorescence examination of diagnostic biopsies is an important adjunct in the classification of amyloid.     

Distinction between envelope antigens of murine xenotropic and ecotropic type C viruses by immunoelectron microscopy.

The indirect ferritin-labelled antibody technique was used to determine the reactivity of an antiserum prepared against the NZB xenotropic virus with three murine xenotropic viruses, a feline xenotropic virus and a murine ecotropic virus. The envelope antigens of the xenotropic type C viruses isolated from the NZB, NIH Swiss and C57L mice were tagged with ferritin. The feline RD114 virus was not. Gross murine leukaemia virus was tagged, but only at high serum concentrations. The cross-reactivity titre of Gross virus to anti-NZB serum was removed by a serum dilution which was still reactive to xenotropic viruses. This difference in reactivity titres between a xenotropic and an ecotropic virus was sufficient to distinguish one from the other in doubly infected cultures. Specific tagging of membrances of cells infected by xenotropic virus was also observed.     

Demonstration of thymus-leukemia (TL) antigens on mitochondria of lymphoid cells by immunoelectron microscopy.

The presence of thymus-leukemia (TL) antigens on the surfaces of mitochondria isolated from TL+ cells, e.g., RADA 1 leukemia cells, and TL+ normal thymocytes, has been directly demonstrated by immunoelectron microscopy. Reagents used were TL alloantiserum and a hemocyanin conjugate of rabbit antibody to mouse IgG. No hemocyanin labeling was observed on mitochondria obtained from TL- cells, e.g., thymocytes of TL- strains, splenocytes of either TL+ or TL- strains, and TL- leukemia cells. The concurrence of TL antigens on the plasma and mitochondrial membranes was shown by the ability of intact cells to absorb all reactivity toward mitochondria.     

Localisation of CD10 to biliary canaliculi by immunoelectron microscopical examination.

Common acute lymphoblastic leukaemia antigen (CALLA) was first characterised in lymphoid leukaemic cells. The antigen is present in different stages of lymphoid cell differentiation as well as in subsets of myeloid cells, and further studies have also shown its presence in non-lymphoid tissues. The recent cloning and sequencing of the gene permitted deduction of its amino acid sequence which is identical with the human membrane-associated enzyme, neutral endopeptidase. Strong immunostaining for CALLA was detected in the human liver with a canalicular pattern. Immunoelectron microscopy also confirmed that the antigen was localised only in the area of the bile canaliculi. Although the function of neutral endopeptidase in the canaliculi is unknown, this antigen may prove useful in the study of biliary function and diseases.     

Isoantigen loss in cervical neoplasia. Demonstration by immunofluorescence and immunoperoxidase techniques.

Tissue sections from normal uterine cervix and from uterine cervices with dysplasia, carcinoma in situ, and invasive cancer were studied in 70 instances using immunofluorescence and immunoperoxidase techniques for demonstrating the presence of isoantigens A and B. Isoantigens were demonstrated in normal squamous epithelium. Antigens were lost in foci of invasive cancer, were considerably reduced or lost in areas of classic carcinoma in situ in all cases, and were reduced in most cases of dysplasia. These observations confirm those obtained with the specific red cell adherence test. The results suggest that immunofluorescence and immunoperoxidase techniques are sensitive, reproducible, easily performed methods for demonstrating the loss of isoantigens in cervical neoplasia.     

Localization of immunodominant antigens of the liver fluke Opisthorchis felineus by immunoelectron microscopy

Immune electron microscopy with a panel of monoclonal antibodies to O. felineus antigens and human immune sera from patients was used for localization of the main antigens of adult O. felineus. The immune complexes at the ultrastructural level were visualized by 5-nm colloidal gold. The main antigens recognized by human antibodies and monoclonal antibodies were associated with the tegument, muscles, uterus, gonads, intestine and eggs of the liver fluke. The findings led to the conclusion that the surface structures of liver flukes stimulate a low B-cell immune response. The structures of the excretory-secretory system of the parasite and their products contain a lot of main antigens and induce B-immune response in man.     

Evidence for expression of the C3d receptor of Candida albicans in vitro and in vivo obtained by immunofluorescence and immunoelectron microscopy.

The complement conversion product C3d binds to a receptor on the cell surface of Candida albicans. While the function of this receptor is still uncertain, we investigated whether it is expressed during a murine infection. Rabbit antiserum raised against purified receptor was used in conjunction with immunofluorescence microscopy and immunocolloidal gold electron microscopy to examine kidney tissue and peritoneal lavages from infected mice for receptor expression by C. albicans in vivo. Specificity of the antiserum was indicated by reactivity with purified receptor (55 to 60 kDa) and with a protein of similar molecular mass from whole hyphal extracts in Western blots (immunoblots). In vitro analysis by immunofluorescence microscopy showed that the antiserum reacted with both yeast and pseudohyphal forms of the organism, but reactivity was strongest with pseudohyphae. Immunocolloidal gold electron microscopy of fungal cells from peritoneal lavages revealed intense staining of mother cells of germinative forms, germ tubes, and pseudohyphae. Staining of the mother cells was heaviest at the innermost layers of the cell wall but only scant on the cell surface. In contrast, staining was observed throughout the cell walls of germ tubes and pseudohyphae. In kidney, expression of the C3d receptor was found primarily on the cell walls of hyphae and pseudohyphae, although some staining was observed in the cytoplasm. These data support that the C3d receptor of C. albicans is expressed in vivo.     

Distribution of cell surface antigens in histiocytosis X cells. Quantitative immunoelectron microscopy using monoclonal antibodies

We have shown previously that cells comprising the cutaneous infiltrates of histiocytes X (HXCs), like Langerhans cells (LCs), react with monoclonal anti-T6 antibody. HXCs also react with anti-T4/4b and anti-Ia-like antibodies. To further define the patterns of antibody reactivity in HXCs, we performed immunoelectron microscopy on two clear-cut cases of histiocytosis X using the immunoperoxidase technique. The reaction product of diaminobenzidine, indicating anti-T6 antibody reactivity, was easily detected along cell membranes of HXCs and at sites of possible endocytosis. Anti-T4/4b and anti-Ia antibodies had less cell membrane reactivity. Computerized image analysis aided in discriminating the patterns of anti-T6 antibody in HXCs and from LCs and confirmed that anti-T6 antibody staining was the most intense of the antibodies evaluated. These findings (1) document antigenic similarities and differences between HXCs and LCs on an ultrastructural level, (2) add support to the concept that HXCs are abnormal proliferations of LCs, and (3) demonstrate the association of the cell surface antigen, T6, with apparent endocytotic activity.     

Binding of monoclonal antibodies to glomerular endothelium, slit membranes, and epithelium after in vivo injection. Localization of antigens and bound IgGs by immunoelectron microscopy.

The antigens recognized by seven monoclonal antibodies (MAbs) raised against rat glomerular proteins were localized, and the sites of binding of the MAbs after in vivo injection were determined by immunoelectron microscopy. The antigens were localized in situ by immunoperoxidase and immunogold labeling to different domains and microdomains of the glomerular endothelium and epithelium. 23A recognized an antigen expressed exclusively on the luminal (apical) domain of the endothelium. 5A (anti-podocalyxin) and 26C (anti-DPPIV) recognized antigens expressed on the apical domains of both the endothelium and podocytes. 13A, 14A, 20B (anti-gp330), and 27A recognized antigens restricted to podocytes in the glomerulus. The 13A antigen was present on their basal surface and the 27A and 14A antigens were expressed on both their apical and basal domains. The 14A antigen also was associated with the filtration slit membranes. All these MAbS bound to their antigens after injection in vivo. Those that recognize endothelial antigens were rapidly cleared from the circulation and rapidly disappeared from glomeruli, whereas those that recognize epithelial antigens persisted in the circulation and were detectable in glomeruli for hours or days. The sites of binding of the MAbs differed: 23A and 5A IgG (antipodocalyxin) bound exclusively to the luminal domain of the endothelium, whereas 26C IgG (anti-DPPIV) bound to both the luminal endothelial membrane and the apical and basal domains of podocytes. The MAbs that recognize podocyte antigens bound to different domains of the podocyte plasmalemma: 13A and 27A IgGs to the basal domain, 14A to the slit membranes, and 20B to coated pits on the entire plasma membrane. 27A IgG led to the formation of small subepithelial immune deposits that remained up to 10 days. It is concluded that 1) glomerular membrane proteins vary considerably in their distribution among plasmalemmal domains and microdomains of endothelial and epithelial cells; 2) virtually all structures in the glomerulus and all domains and micro-domains of the endothelium and podocyte are accessible to circulating antibodies; and 3) the fate of immune complexes formed by binding to glomerular components varies with the location of the antigen within the glomerulus, with those that bind to the basal domain and slit membranes of the podocyte persisting longer than the others.     

Localization of the tube precipitin and complement fixation antigens of Coccidioides immitis by immunoelectron microscopy with murine monoclonal antibodies.

The cellular localization of the tube precipitin (TP) and complement fixation (CF) antigens of Coccidioides immitis was examined by immunoelectron microscopy with murine immunoglobulin G1 monoclonal antibodies directed against the TP and CF antigens, respectively. Immunoelectron microscopic analyses of saprobic- and parasitic-phase cells showed that the TP antigen is present at a high concentration within the inner cell wall layer and along the plasma membrane. The antigen was also detected, at a lesser concentration, within cytoplasmic vacuoles. In contrast to the predominant localization of the TP antigen in the cell walls, the CF antigen residues primarily within the cytoplasm, where it appears to be dispersed throughout the cytoplasm rather than associated with a specific cytoplasmic organelle. A sparse amount of the CF antigen within the inner cell walls was also demonstrable. The localization of the TP and CF antigens throughout the morphogenetic phases of C. immitis has important implications in antigen production and in analyses of host response in coccidioidomycosis.     

Detection of HLA-D-region-associated antigens on the surface of adult T-cell leukemia virus particles by immunoelectron microscopy

To search for lymphocyte marker antigens on the surface of human T-cell leukemia virus (HTLV), an immunoelectron microscopic study was performed on a HTLV-producing human T-cell line, MT-2, using monoclonal antibodies, such as anti-Leu-1, -Leu-2b, -Leu-3a, -Leu-5, -Leu-10 and -HLA-DR and OKIal. The reactivity of each antibody with MT-2 cells was tested by the immunoperoxidase method at the light microscopic level. OKIal, anti-HLA-DR and -Leu-10 gave positive results. At the ultrastructural level, the surface of HTLV as well as the plasma membranes of MT-2 cells were labeled with ferritin by the monoclonal antibodies OKIal, anti-HLA-DR and -Leu-10, but not by anti-Leu-1 and -Leu-3a. These findings suggest that HLA-D region -associated antigens are common antigenic determinants shared by the surface of HTLV and the plasma membranes of MT-2 cells. These antigens on the virus surface are probably picked up selectively from the plasma membranes and may play an important role in the interaction of HTLV and target T-cells.     

Detection of chicken anaemia agent in chickens by immunofluorescence and immunoperoxidase staining

An immunofluorescent method for the detection of CAA antigens in impression smears and cryostat sections of bone marrow and thymus tissue from infected birds is described. CAA antigens were detected using both rabbit and chicken antisera to the virus. A protocol for the demonstration of CAA antigens in formalin-fixed, paraffin-embedded thymus tissue, using a biotin-streptavidin peroxidase detection system, was also developed. Treatment of tissue sections with eleven protease solutions, prior to application of primary antibody, was evaluated. Only protease types III and XIV were successful in unmasking CAA antigens while preserving good tissue morphology with no non-specific background staining.     

Localization of a murine oncornavirus 15,000-dalton virion protein on the membrane of neoplastic cells: analysis by immunofluorescence and immunoelectron microscopy.

A protein analogous to a 15,000-dalton structural protein of Rauscher murine oncornavirus has been localized on cell membranes of Rauscher erythroblastosis and CC57BR murine lymphosarcoma cells by use of monospecific antiserum in conjunction with immunofluorescence and immunoelectron microscopy techniques. Antigenic determinants of this protein were present on the membranes of erythroblastosis and lymphosarcoma cells, but were absent from the membrane of budding or mature virions. Both typespecific and group-specific determinants of the protein were accessible to antibodies reacting at the cell surface. This viral protein, expressed on the cell membrane, appears to correspond to previously described cell surface antigens of virus-induced murine leukemias.     

Detection of carcinoembryonic antigen in tissue sections by immunoperoxidase.

A triple-bridge, indirect, immunoperoxidase method for detecting and localizing carcinoembryonic antigen (CEA) in tissue sections is described. By this technique, a cell-surface localization of CEA in colonic carcinoma and ovarian mucinous cystadenocarcinoma cells could be visualized. In the case of the colonic cancer, both the tumor from the descending colon and a metastasis to the skin gave positive peroxidase reactions for CEA. This immunocytochemical method for demonstrating the presence of CEA functioned in both frozen, ethanol-fixed and formalin-fixed, paraffin-embedded tissues, thus making it applicable for use with tissue sections conventionally prepared for light microscopy.     

Immunocytochemical reaction of Ca1 and HMFG2 monoclonal antibodies with cells from serous effusions.

The Ca1 antibody was used in an alkaline phosphatase immunocytochemical method on cells obtained from 150 specimens of pleural and ascitic fluids. The results were compared with the routine cytology report based on the light microscopical appearances. The Ca1 antibody identified tumour cells in 51 of 57 specimens with malignant cells. The exceptions were four small cell carcinomas, one malignant lymphoma, and one adenocarcinoma. A further seven specimens reported as containing atypical cells but without conclusive evidence of malignancy were Ca1 positive. The Ca1 antibody did not give a positive reaction with benign mesothelial cells. Similar results were obtained with the HMFG2 antibody and malignant cells, but in eight of 18 benign effusions it reacted with mesothelial cells.