Immunoelectron microscopic localization of herpes simplex virus antigens in infected cells using the unlabeled antibody-enzyme method.

Subcellular localization of viral antigens was demonstrated during viral morphogenesis using herpes simplex virus type 1 (HSV-1) infected monolayers of rabbit cornea cells. The localization was done by immunoelectron microscopy employing the peroxidase-antiperoxidase (PAP) immunocytochemical technique and the postembedding staining method. The localization of viral antigens was followed at time intervals during infection from 2 to 19 hr. After exposure of sections to either polyspecific antibodies against total HSV-1 antigens or monospecific antibodies against HSV-1 antigen No. 8, specific immunological reaction products were identified both in the cytoplasm and nucleus after 2 hr. The distribution and quantity of reaction products varied in the infected cells during the viral morphogenesis. The present results on the subcellular distribution of the HSV-1 antigens are related to current biochemical findings.     

Immunocytochemistry with osmium-fixed tissue. I. Light microscopic localization of growth hormone and prolactin with the unlabeled antibody-enzyme method.

Growth hormone and prolactin were localized on thin plastic sections of rat anterior pituitary gland and mammosomatotropic tumor MtTW15 that were fixed with osmium tetroxide (alone,mixed with aldehydes, or after aldehydes). Intense immunocytochemical staining for both antigens was obtained after plastic was removed from sections with an alcoholic solution of sodium hydroxide. The results indicated that antigenic determinants of rat prolactin and growth hormone were not completely destroyed or inactivated by fixation with osmium and embedment in epoxy resin, and that removal of the polymerized epoxy resin was necessary to obtain light microscopic postembedding immunocytochemical staining of these antigens. The results also demonstrated that tissues which have been conventionally processed for morphological evaluation by electron microscopy may be suitable for postembedding immunocytochemical staining of some antigens for light microscopy.     

Ultrastructural immunocytochemical localization of vitellogenin in the fat body of the blowfly,Calliphora vicina Rob.-Desv. (erythrocephala Meig.) by use of the unlabeled antibody-enzyme method

Summary The unlabeled antibody-enzyme method was used to demonstrate ultrastructurally the specific localization of vitellogenin in the fat body of Calliphora. In control flies the binding sites to vitellogenin were localized in secretory granules situated in the Golgi complex, and in larger bodies named composite secretory granules. These composite granules appear to be formed when a part of a Golgi complex containing secretory granules and a number of small vesicles become surrounded by a common membrane. Ovariectomized flies, which apparently do not produce secretory granules, exhibited no immunocytochemical staining. Ovariectomized flies in which the administration of ecdysterone induced formation of secretory granules, also revealed specific staining on these granules. This is the first ultrastructural evidence of: (a) the specific localization of vitellogenin in secretory granules of the fat body of an insect; (b) the relationship between the presence of the ovary, and of ecdysterone, and the synthesis of vitellogenin by the fat body.     

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.     

Ultrastructural studies of H-1 parvovirus replication : III. Intracellular localization of viral antigens with immunocytochrome c

Immunoelectron microscopy with cytochrome c conjugated anti-H-1 IgG was used to localize antigens of the parvovirus H-1 within synchronized human NB cells. Since glutaraldehyde destroyed H-1 antigenicity, a fixative containing formaldehyde was developed which preserved both cellular ultrastructure and antigenic function. The earliest H-1 specific staining occurred on the heterochromatin bordering the nuclear envelope at 8 h post infection (p.i.). At 10 h p.i., labeling was found on the chromatin associated with the nucleolar surface and tufts of heterochromatin distributed throughout the nucleoplasm. Except for this H-1 labeling, the chromatin appeared indistinguishable from that of uninfected cells. By 12 h p.i., however, coinciding with the abrupt rise in synthesis of H-1 hemagglutinin and infectious virus, H-1 labeled intranuclear chromatin had condensed and migrated toward the nuclear membrane. Also, trabeculae of intranuclear chromatin were tagged with anti-H-1 conjugate as contraction of nucleolar chromatin and disintegration of nucleolar ultrastructure began. Condensation of the nucleolar associated chromatin and nuclear heterochromatin appeared complete by 18–36 h p.i. when thick zones of this H-1 labeled material were observed at the nucleolar and nuclear periphery. Our results indicate that the binding of unassembled H-1 proteins to specific regions of chromatin is associated with their condensation and margination, resulting in early nucleolar destruction and subsequent nuclear damage during H-1 infection.     

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.     

Electron-microscopic localization of baboon acrosomal antigens using antisperm monoclonal antibody.

A sperm antigen corresponding to baboon sperm monoclonal antibody 1A9 was localized in the testis and ejaculated sperm in this animal, using the immunofluorescence technique and immunogold labelling. Immunohistochemical studies of the baboon testis showed that the antigenic determinant was localized in the late spermatid cells and spermatozoa close to the seminiferous tubules. Immunofluorescence studies indicate that the protein was localized on the acrosome region of ejaculated baboon sperm. At the electron-microscopic level, gold particles indicative of the presence of this determinant recognized by 1A9 monoclonal antibody were detected on the inner acrosomal region of ejaculated baboon sperm.     

A modification of the unlabeled antibody enzyme method using heterologous antisera for the light microscopic and ultrastructural localization of insulin, glucagon and growth hormone.

The requirement of using homologous antisera (primary antiserum and peroxidase-antiperoxidase (PAP) complex raised in the same species) in the unlabeled antibody enzyme method has been investigated at the light and electron microscopic level using the localization of insulin, glucagon and growth hormone as model systems. Optimum immunocytochemical staining for all three antigens was observed when sheep or goat antirabbit gamma-globulin (S-ARgammaG or G-ARgammaG) were used to couple rabbit peroxidase-antiperoxidase complex with either guinea pig antisera to insulin (GP-AIS) or glucagon (GP-AGS), or monkey antisera to rat growth hormone (M-ARGH). The cross-reactivity between S-ARgammaG or G-ARgammaG and immunoglobulins in these primary antisera were substantiated by immunoelectrophoresis and radioimmunoassay. S-ARgammaG was shown to produce precipitation arcs with GP-AIS and M-ARGH that were similar to those seen when the latter were reacted with rabbit antiguinea pig gamma-globulin antiserum and goat antimonkey gamma-globulin antiserum, respectively. Radioimmunoassay results revealed that immunoprecipitation of 6-10% as compared to homologous antisera controls yielded excellent staining localization when S-ARgammaG was used for immunocytochemistry. Thus, heterologous antisera (primary antiserum and PAP complex raised in different species) may be used in the unlabeled antibody enzyme method as long as the coupling antiserum shows cross-reactivity with immunoglobulins of the primary antiserum and the PAP complex.     

Ultrastructural localization of nuclear antigens during interphase in mouse 3T3 fibroblasts

Thin sections of mouse 3T3 fibroblast nuclei labelled by immunoperoxidase with anti-nuclear antibodies I1, PI1, PI2, anti-peripherin, -lamin, and -centromere have been examined in the electron microscope. Staining was compared with the corresponding immunofluorescence labelling patterns, and was correlated with nuclear ultrastructure in conventionally fixed and uranyl-lead stained samples and in unlabelled immunoperoxidase controls. Peripherin was detected at the nuclear rim in a band broader and more irregular than the lamins/lamina. The peripheral components of PI1 and PI2 appear to be localized at nuclear pores and the nuclear envelope, respectively. The internal component of PI1 staining consisted of irregular patches and strands in the nucleoplasm, closely resembling snRNP staining as reported by others. Internal P12 labelling consisted of spots distributed apparently at random in interchromatinic regions. The spots resembled labelling by antibody I1, but were fewer and more irregular in size. Neither the PI2 nor the I1 spots were centromere associated, nor could they be correlated with specific interchromatinic structures in conventional preparations and in unlabelled controls. The results support the hypothesis that the nucleus is segregated into function-specific domains, distinguished by morphology and (or) composition from surrounding regions of the nucleus.     

Ultrastructural localization of acidic glycoconjugates with the low iron diamine method

The present study has applied the low iron diamine (LID) method at the ultrastructural level to demonstrate acid glycoconjugates. We have examined rat epiphyseal cartilage, human bone marrow, rat tracheal glands, and mouse sublingual glands stained with LID prior to embedment. The LID staining appeared to require postosmication for adequate visualization at the electron microscope level. Thiocarbohydrazide-silver proteinate (TCH-SP) staining of thin sections variably enhanced LID reactive sites. LID-TCH-SP stained carboxyl and sulfate groups of glycosaminoglycans in the extracellular cartilage matrix, secretory granules, and expanded Golgi saccules of chondrocytes. In human bone marrow, LID-TCH-SP variably stained the cytoplasmic granules, known to contain sulfated glycosaminoglycans, and the external surface of the plasma membrane of leukocytes. Moderately strong LID staining was observed in secretory granules in mucous tubules of rat tracheal glands, known to contain sulfated glycoproteins, and in acinar cells of mouse sublingual glands, known to contain a sialoglycoprotein. The lack of sulfated glycoconjugates in acinar cells of the mouse sublingual gland was confirmed by their failure to stain with the high iron diamine method. Thus these studies indicate that the LID and LID-TCH-SP methods are useful for the ultrastructural localization of carboxylated and sulfated glycoconjugates in extracellular and intracellular sites.     

Ultrastructural localization of bcl-2 protein.

Previous cell subfractionation studies have indicated that bcl-2 is an inner mitochondrial membrane protein. We have sought to determine the ultrastructural localization of bcl-2 protein in lymphoma and breast carcinoma cell lines and biopsy material known to overexpress bcl-2 using immunoelectron microscopy. To avoid the possibility of processing artifacts, samples were prepared by three different methods: progressive lowering of temperature, cryosectioning, and freeze-substitution. In all instances the labeling of bcl-2 protein was relatively weak but the distribution the same. In both lymphoma and breast carcinoma tissues, bcl-2 protein was detected on the periphery of mitochondria: little labeling of either the mitochondrial matrix or cristae could be detected. Labeling was also detected on the perinuclear membrane and throughout the cytoplasm, as also indicated by confocal microscopy. These data therefore indicate that bcl-2 protein can be detected at several intracellular sites and that at the likely functional destination, the mitochondria, there appears to be, contrary to expectations, a preferential association with the outer membrane.     

Ultrastructural localization of neurophysin-like immunoreactivity in the rat posterior pituitary. An alternative method using frozen-dried tissue fixed with OsO4 vapor.

Electron microscopic identification of elements containing neurophysin-like immunoreactivity can be accomplished in rat posterior pituitary that has been frozen-dried and fixed with OsO4 vapor. Alternating serial ultrathin sections are placed on grids and glass slides. The sections on the slides are stained for neurophysin using immunofluorescence histochemistry, and the resultant images are superimposed on electron micrographs from the adjacent sections. The method provides several advantages for the localization of neuropeptide immunoreactivity in nervous tissue.     

Ultrastructural localization of an extracellular protease in Pseudomonas fragi by using the peroxidase-antiperoxidase reaction.

An extracellular protease, which previously has been found to correlate with the appearance of bleblike evaginations on the cell wall of Pseudomonas fragi ATCC 4973, was purified 38-fold by ammonium sulfate precipitation and Sephadex chromatography to yield a single band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Polyclonal rabbit antiserum raised against the purified enzyme had an enzyme-linked immunosorbent assay titer of 4 X 10(7). The peroxidase antiperoxidase method was used to localize the neutral protease in P. fragi at the ultrastructural level. Electron microscopy of cell sections of this organism revealed that high concentrations of positive immunoperoxidase reaction product were located near the cell wall, whereas control sections stained with preimmune or heterologous serum did not show similar deposits to be present. These results are consistent with the hypothesis that blebs appearing on P. fragi contain high concentrations of neutral protease.     

Ultrastructural localization of heavy metals by a modified sulfide-silver method

Summary An ultrastructural modification and application of the sulfide-silver method for cell suspensions and ultrathin sections of tissue has been described using glutaraldehyde saturated with hydrogen sulfide as a fixative. When, after physical development in a modified sulfide-silver developer, silver grains appeared in fully treated cells and sections but neither in ungassed but developed controls, nor in gassed but non-developed sections, they were thought to give the sites of heavy metals. A positive reaction for heavy metals has been described to occur in the secretion granules of pancreatic -cells of dog, cat, Chinese hamster, rabbit, and rat, as well as the fish Cottus quadricornis. On isolated cells a positive reaction was obtained in the fungus Cryptococcus neoformans, on the surface of all rat peritoneal cells and cattle blood leucocytes, as well as in erythrocytes and in the specific granules of rat mast cells and rat and cattle eosinophils.The limitations and critical methodological points of the procedure are given, as well as the precautions needed in the interpretation of the results.This work has been supported by grants from the Swedish Medical Research Council (project No. 12X-718-01) and from the Medical Faculty, the University of Umeå.