Journal of Histochemistry & Cytochemistry

Howard, R J; Battye, F L; Mitchell, G F

doi: 10.1177/27.4.87413pmid: 87413

Red cells from Plasmodium berghei infected mouse blood can be sorted on the basis of their DNA content with the bisbenzimidazole dye 33258 Hoechst. The optimal conditions for dye uptake have been established and with these conditions uninfected cells are nonfluorescent and can be completely separated from infected cells which exhibit fluorescence in almost direct proportion to the number of parasite nuclei (i.e. DNA) they contain. The number of fluorescent cells detected and their fluorescence intensity is shown to be dependent on the dye concentration and the incubation medium being used. At least a proportion of the infected cells sorted from each fluorescence peak in the cell distribution retain their infectivity in vivo with some, but not all, conditions of labeling. This technique is being used to separate minor cell populations from infected blood for biochemical and immunochemical analyses and to screen human samples for malaria infected cells.

Christie, K N; Stoward, P J

doi: 10.1177/27.4.376691pmid: 376691

Catalase has been localized immunocytochemically with anti-bovine catalase in long thin filament structures in aerobic type I fibers in the skeletal muscles of normal and genetically dystrophic hamsters. The filaments range in length from 1 to 60 micron, are orientated regularly along the long axis of the fibers, and also seem to surround and project from muscle nuclei. The enzyme thus appears to be more prominent in the sarcoplasmic reticulum than in peroxisomes, and in this situation is suitably placed for destroying toxic hydrogen peroxide which may be continously generated in aerobic fibers.

Spicer, S S; Stoward, P J; Tashian, R E

doi: 10.1177/27.4.109495pmid: 109495

Carbonic anhydrase has been localized with an immunoenzyme bridge technique in the following sites in paraffin sections of fixed rodent tissues: gastric parietal cells, the brush border of enterocytes in the small intestine, superficial nongoblet cells of the colon, selective segments of the nephron, glial cells, erythrocytes and adipose cells. Immunocytochemical localizations of carbonic anhydrase isozymes I and II in different histologic sites, by means of affinity column purified antibodies, agreed with the distribution of these enzymes in the various sites, as indicated by immunologic assays. The immunocytochemical results are compared with those reported for the cobalt-bicarbonate cytochemical method and with biochemical knowledge of the occurence of carbonic anhydrase.

Mason, D Y; Sammons, R E

doi: 10.1177/27.4.109496pmid: 109496

A two stage immunohistological technique (the "labeled antigen" procedure) has been assessed for the detection of a variety of human and animal cytoplasmic constituents in tissue sections. In this method specific antiserum is followed by antigen complexed to horseradish peroxidase or to alkaline phosphatase. The primary antibody acts bivalently, linking the labeled antigen to antigen in the tissue section. The major advantage of this technique is that nonantigen specific antibody in the primary antiserum cannot cause nonspecific staining since it has no affinity for the antigen:enzyme complex. Consequently the specificity of the reaction is assured, background staining is minimized and the total staining time (from wax section to mounted slide) can be reduced to as little as 30 min. Further advantages include the possibility of labeling Ig allotypes and the high efficiency of enzyme utilization. Covalent human IgG:horseradish peroxidase complexes can also be used in a triple sandwich in conjunction with human anti-viral or autoimmune antibodies.

Ripoche, M A; Bernard, O J; Bennett, D

doi: 10.1177/27.4.36428pmid: 36428

Several peroxidase-Ig conjugates were applied to sections of fixed mouse tissue. When the peroxidase staining reaction was done at pH 4.5 instead of pH 7.4, a striking reaction on nuclear membrane, chromatin, or chromosomes was observed. This staining was prevented by pretreatment of sections with DNase but not with RNase or after acid elution of histones. It is suggested that at acid pH a redistribution and binding DNA of oxidized chromogen or of a chromogen-conjugate complex to DNA may account for the results observed.

Vogel, Z; Towbin, M; Daniels, M P

doi: 10.1177/27.4.376692pmid: 376692

A method is presented for the efficient conjugation of horseradish peroxidase to alpha-bungarotoxin. The 1:1 molar conjugate obtained is purified to completion by gel filtration on Sephadex G-100, followed by ion exchange chromatography on CM-Sephadex. The conjugate retains half of the activity of unmodified horseradish peroxidase and binds effectively to the nicotinic acetylcholine receptor of muscle. The conjugate is proven to be useful reagent for the histochemical staining of the receptor on muscle fibers for light and electron microscopy.

Douglas, W H; Hitchcock, K R

doi: 10.1177/27.4.582179pmid: 582179

Organotypic cultures, established from enzymatically dispersed day 19 fetal rat lung, are comprised primarily of cells which are morphologically similar to type II alveolar pneumonocytes, the cells involved in surfactant synthesis. To further characterize these cultures, the nonspecific esterase pool was examined to determine if these cultures contained certain nonspecific esterases previously shown to be enzyme markers for the surfactant system. The results of biochemical, electrophoretic and cytochemical studies indicate that these organotypic cultures contain the same nonspecific esterases already demonstrated in surface active fractions derived from rat and mouse lung homogenates and pulmonary lavage fluid. As in whole lung, the major site of esterase activity in the organotypic cultures is the type II cell lamellar body, the putative site of surfactant synthesis and storage. These findings support the concept that the organotypic cultures derived from fetal rat lung are comprised predominantly of type II cells which retain surfactant associated functions in vitro.

Le Bouteiller, P P; Toullet, F; Righenzi, S; Voisin, G A

doi: 10.1177/27.4.376693pmid: 376693

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.

Baskin, D G; Erlandsen, S L; Parsons, J A

doi: 10.1177/27.4.109497pmid: 109497

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.

Sannes, P L; Katsuyama, T; Spicer, S S

doi: 10.1177/27.4.448061pmid: 448061

The matrix of some mitochondria in gastric parietal cells of rat and guinea pig evidenced affinity for the high iron diamine method which localizes sulfated complex carbohydrates selectively by light and electron microscopy. Such staining has not been observed elsewhere in the stomach. The high iron diamine reactive mitochondria about equaled in number those which were unreactive, and the two groups were indistinguishable morphologically. The distinction was not apparent either when mitochondria were stained by other cytochemical procedures including dialyzed iron for acidic complex carbohydrates, 3-3' diaminobenzidine-H2O2 at pH 6.0 for cytochrome oxidase, and Kominick's pyroantimonate osmium tetroxide for antimonate precipitable cations. The dialyzed iron method stained acid glycoconjugates in the outer intermembrane space in parietal cell mitochondria. These mitochondria stained more strongly with dialyzed iron than have any others examined heretofore with this method and comprised the only reactive mitochondria in the stomach. Parietal cell mitochondria also stained strongly for cytochrome oxidase but those of other gastric cells failed to evidence this reactivity.