Journal of Histochemistry & Cytochemistry

Takamiya, H; Batsford, S; Vogt, A

doi: 10.1177/28.10.6158534pmid: 6158534

A method is described for performing postembedding staining of protein (immunoglobulin) antigen embedded in styrene-methacrylate resin. Fixation of specimens in a combination of 4% paraformaldehyde and 0.2% picric acid and washing in buffer containing 7% sucrose, followed by abrupt dehydration with absolute acetone in the cold preserved the antigenicity, although in a masked form. The masked antigenicity could be reexposed by treatment with nonspecific protease. Staining with fluorescent-, peroxidase-, or ferritin-labeled antibodies on semi- and ultrathin sections resulted in specific localization of the antigen. We applied this technique to the localization of rabbit immunoglobulin in specimens of renal tissue obtained from rats with anti-glomerular basement membrane nephritis; we also localized human IgG in a renal biopsy specimen. The prerequisites for recovery of antigenicity are such that preservation of tissue structure at the light microscopic level is good, but relatively poor at the electron microscopic level.

Blackett, N M; Parry, D M; Baker, J R

doi: 10.1177/28.10.7419897pmid: 7419897

Analysis of autoradiographs at the electron microscope level requires special procedures, since the size of the radioactive structures visualized are comparable to the range of the radioactive decay particles emitted. Quantitative analysis in these circumstances requires that the sizes, shapes, and juxtaposition of the various structures be taken into account in relation to the range distribution of silver grains, produced by the decays, from a point source for the particular isotope and autoradiographic conditions employed. We present the distribution of silver grains about a point source for the four electron capture isotopes 51Cr, 55Fe, 111In, and 125I. Thin radioactive line sources were constructed and the distribution of autoradiographic grains measured. The grain distributions are discussed in relation to the number of particles per disintegration and their energy and range. A simple calculation enables these line source distributions to be converted into point source distributions, which can then be used for whichever method of quantitative analysis is considered appropriate for a particular problem. An outline is given of some of the more important aspects of various methods of analysis.

Bentfeld-Barker, M E; Bainton, D F

doi: 10.1177/28.10.7419898pmid: 7419898

Basophils and mast cells possess large metachromatically staining granules which contain sulfated glycosaminoglycans as well as vasoactive compounds. To determine whether these granules might also have lysosomal properties, we used electron microscopy and cytochemistry to localize arylsulfatase B in rat basophils and mast cells. In basophils of bone marrow, enzymatic reaction product was consistently seen in many, but not all, of the basophil granules. In some cells, the enzyme could also be demonstrated in the Golgi region, restricted to a single cisterna and small vesicles. It was never seen in rough endoplasmic reticulum (RER), although the paucity of cells made adequate sampling difficult. In mast cells of bone marrow and the peritoneal cavity, enzymatic reaction product was consistently found in some cytoplasmic granules of varying sizes and shapes where it characteristically rimmed the periphery of the granule just beneath the limiting membrane. It should be emphasized, however, that the majority of granules were not reactive. Reaction product could also be found occasionally in segments of RER, and in the Golgi region with a distribution similar to that of the basophil. The presence of lysosomal arylsulfatase in granules of developing basophils in bone marrow suggests that some basophil granules, like those of neutrophils, eosinophils, and monocytes are primary lysosomes. Some mast cell granules also contain this lysosomal enzyme, although it is not clear from the present data whether these granules are primary or secondary lysosomes.

Teichman, J S; Krick, T P; Nettleton, G S

doi: 10.1177/28.10.6158535pmid: 6158535

The Feulgen reaction is used for cytophotometric quantitation of nuclear DNA. Schiff's reagents used in the Feulgen reaction usually are prepared from basic fuchsin, a variable mixture of four triaminotriphenylmethane analogs. The effect of the several fuchsin analogs on the quality of Schiff's staining of hydrolyzed DNA is not known. In this investigation Schiff's reagents prepared from relatively pure fuchsin analogs were used to determine whether different fuchsin analogs affect the absorbance of the Schiff's reagent-DNA complexes formed in solution. It has been determined that the complex formed by pararosaniline-Schiff's reagent and hydrolyzed DNA exhibits lower absorption than do corresponding complexes formed by Schiff's reagents prepared from magenta II or from new fuchsin.

Parmley, R T; Eguchi, M; Spicer, S S; Alvarez, C J; Austin, R L

doi: 10.1177/28.10.7419899pmid: 7419899

The subcellular route of incorporation of complex carbohydrates into rabbit heterophil primary granules and their subsequent intragranular distribution during granule maturation were studied with ultrastructural, cytochemical, and radioautographic methods. High iron diamine (HID) staining of sulfated glycoconjugates in primary granules was partially diminished after treatment with chondroitinase ABC or after removal of N-sulfate groups with nitrous acid, but was not altered by exposure to hyaluronidase, trypsin, or HCl. Subsequent thiocarbohydrazide-silver proteinate (TCH-SP) straining of thin sections increased the density of the HID reaction product. Golgi-derived spherules and very immature morular granules stained weakly with HID-TCH-SP and labeled intensely after a 10 min incubation with 35SO4. After a 60 min 35SO4 pulse and a 60 min chase, an increase in radiolabeling was observed in granules with HID stained, fused morular material, and some labeling was present in more mature rim stained granules. Fully mature granules lacked HID or HID-TCH-SP staining, but contained most of the 35SO4 labels after a 60 min pulse and 18 hr chase in vitro. Periodate-thiocarbohydrazide-silver proteinate (PA-TCH-SP) staining of unosmicated thin sections localized vicinal glycol-containing complex carbohydrates in Golgi-associated small vesicles. These vesicles lacked HID-TCH-SP staining and apparently contained neutral glycoprotein. They frequently bordered, in a rosette arrangement, the immature morular granules, but not the more mature primary granules. The PA-TCH-SP method localized complex carbohydrates in the rim of granules precursors and enclosed a spherule or morula, but failed to stain the sulfate-containing material in the morulas or spherules. PA-TCH-SP reactivity was diffusely distributed in moderately mature granules and was decreased in fully mature granules. These results indicate that heterophil primary granule contain several complex carbohydrates including O-sulfated and N-sulfated glycosaminoglycans, as well as vicinal glycol-containing glycoproteins. These complex carbohydrates are transported to immature primary granules by different Golgi-derived organelles. The complex carbohydrates are subsequently distributed differently within primary granules and become masked to staining as the granule matures.

Nenci, I; Dandliker, W B; Meyers, C Y; Marchetti, E; Marzola, A; Fabris, G

doi: 10.1177/28.10.7419900pmid: 7419900

A recently synthesized fluorescein-labeled estrogen (17FE, 1-(N)-fluoresceinyl-estrone-thiosemicarbazone) interacts with estrogen-target cells like the native hormone and visualizes the uptake, transport, and distribution of estrogen in intact target cells. Moreover, estrogen binding sites are traced by 17FE in cryostat sections of estrogen target tissues as well. Cell and tissue 17FE binding sites fulfill the accepted criteria for specific estrogen receptors (finite binding capacity, high affinity, steroid and tissue specificity). This fluorescent probe allows estrogen receptors to be studied in a wide variety of cell and tissue preparations under varying conditions of physiologic and pathophysiologic interest.

Van Ewijk, W; Rouse, R V; Weissman, I L

doi: 10.1177/28.10.6999083pmid: 6999083

Antigens coded for by the major histocompatibility complex (MHC) are differentially expressed in the mouse thymus. Immunoperoxidase studies of frozen thymus sections incubated with monoclonal (hybridoma) anti-I-Ak antibodies revealed a dendritic straining pattern in the cortex and a confluent staining pattern in the medulla. Serial sections incubated with monoclonal anti-H-2Kk antibodies showed that H-2Kk antigens were only present at detectable levels in the medulla. Microenvironments expressing H-2Kk antigens also expressed I-Ak antigens. In cortico-medullary regions, relatively large MHC-negative areas were found. These areas appeared to connect to perivascular spaces surrounding blood vessels. Using a new postfixation labeling method for the detection of cell surface associated antigens on cells of the lymphoid system in situ, we have characterized the nature of MHC positive cell types at the ultrastructural level. These studies show that epithelial-reticular cells are the major MHC positive elements in the thymus. Lymphocytes in the medulla and in cortico-medullary bounderies are also MHC positive, however, lymphocytes in the cortex were not detectably labeled. These findings support the contention that epithelial-reticular cells are involved in the H2-restriction process during T cell maturation.

Ackerman, G A; Wolken, K W; Gelder, F B

doi: 10.1177/28.10.6775025pmid: 6775025

The cholera toxin-colloidal gold-labeled IgG-F(ab')2 anticholera toxin ultrastructural immunocytochemical procedure was used for the localization of GM1 monosialoganglioside on the surface of human blood cells. The number of gold particles per micron of cell surface was counted and the data subjected to statistical analysis. Cholera toxin (CT) binding characteristics assessed in several subjects showed consistent labeling patterns for the various hemic cells, although some quantitative differences were noted in surface labeling densities between subjects. Neutrophils were invariably the most heavily labeled of the hemic cells, while lymphocytes, erythrocytes, and platelets exhibited only limited CT labeling. Exposure of hemic cells to neuraminidase induced a major increase in surface CT labeling that proved to be directly related to cell type and differed in many respects with the CT labeling pattern noted in nonenzyme treated cells. Newly exposed CT binding sites attributed to "masked" GM1 and/or to neuraminidase-transformed GD1a or GT1 gangliosides, showed that the number of new binding sites were nearly twice as abundant on platelet and monocyte sufaces as on the surfaces of neutrophil, lymphocyte, and erythrocyte populations. However, ratios of new CT binding sites to those normally available for CT binding were approximately 10:1 for erythrocytes, approximately 3--7:1 for lymphocytes, monocytes, and platelets, and approximately 1:1 for the neutrophil group. Exogenous GM1 was incorporated into the cell surface of the hemic cells in a differential manner. Platelets showed a dramatic increase in surface CT labeling, viz. approximately 12- to 20-fold, compared to that of other hemic cells; however, neutrophil and erythrocyte GM1 uptake was limited. Our studies have demonstrated that distinct differences exist in the extent of surface CT labeling of the various types of blood cells. They further indicated that the ability of the cell surface to incorporate exogenous GM1 may represent a differential expression of the physiochemical properties of the surface of the individual cell types.

Tanaka, T; Gresik, E W; Michelakis, A M; Barka, T

doi: 10.1177/28.10.6999084pmid: 6999084

By using antibodies against highly purified submandibular gland renin, renin was localized immunocytochemically at the light and electron microscopic level in the submandibular glands and kidneys of adult male SWR/J and C57BL/6J mice. In accord with the data of Wilson et al. (Proc Natl Acad Sci USA 75:1185, 1977), renin was demonstrable only in the submandibular glands of SWR/J mice (high strain), where it was confined to the secretory granules of the granular convoluted tubules. In the kidneys of both strains, renin was confined to epithelioid cells of the juxtaglomerular apparatus. Electron microscopically immunostaining was restricted to the granules of the juxtaglomerular epitheliod cells. Morphometric analyses suggested that the kidney of the C57BL/6J mice contained more immunoreactive complexes per unit volume of cortex than SWR/J mice kidney. The data indicate that submandibular gland renin cross-reacts with kidney renin, but that genetic controls of these polypeptides in the two organs are independent.

Muther, T F; Friedland, B R

doi: 10.1177/28.10.6775026pmid: 6775026

The high binding affinity of acetazolamide for carbonic anhydrase (K1 congruent to 10(-8) M) was employed to demonstrate the distribution of the enzyme in the rabbit ciliary body by incubating the tissue with 3H-acetazolamide (1.5 Ci/mmol). Specificity of binding was ascertained by displacing 3H-acetazolamide with a high concentration of unlabeled ethoxzolamide (K1 congruent to 10(-9) M). Wedges of the globe anterior to the ora serrata were incubated in bicarbonate buffered physiological saline, 95% O2/5% CO2, at 0 degrees C for 2 hr with either 3H-acetazolamide (0.2 microM), 3H-acetazolamide and unlabeled ethoxzolamide (100 microM), or physiological saline alone. They were then washed for 2 hr in fresh physiological saline and processed for autoradiography. The autoradiographs showed the label localized in both pigmented and nonpigmented layers of ciliary body epithelium in the pars plicata and in the iridial processes. The epithelia of both crests and troughs showed localization of label. In contrast, no concentration of label was found in the stroma of the ciliary body, including vascular endothelium, and in the epithelia of the pars plana. In sections that were incubated with 3H-acetazolamide in the presence of an excess of unlabeled ethoxzolamide, no localization of label occurred. These findings suggest that the epithelia of the pars plicata, but not those of the pars plana, contain carbonic anhydrase. This is consistent with hypotheses restricting aqueous humor formation to the pars plicata.