Journal of Histochemistry & Cytochemistry

Roth, J

doi: 10.1177/31.8.6190857pmid: 6190857

A method is described for the electron microscopic detection of lectin-binding sites in different cellular compartments and extracellular structures that uses thin sections from resin-embedded tissues. Various lectins (Ricinus communis lectin I and II, peanut lectin, Lotus tetragonolobus lectin, Ulex europeus lectin I, Lens culinaris lectin, Helix pomatia lectin, and soybean lectin) were bound to particles of colloidal gold and used for direct staining of thin sections or glycoprotein--gold complexes were prepared and applied in an indirect technique (concanavalin A and horseradish peroxidase--gold complex; wheat germ lectin and ovomucoid--gold complex). The details for preparation of such complexes from 14 nm gold particles are reported. The conditions of tissue processing that gave satisfactory staining results and good fine structure preservation were mild aldehyde fixation without osmification and low temperature embedding with the hydrophilic resin Lowicryl K4M. None of the so-called etching procedures was necessary prior to labeling of Lowicryl K4M thin sections. Examples of the use of this approach for detection of glycoconjugates in the rough endoplasmic reticulum, Golgi apparatus, and mucin of intestinal goblet cells as well as plasma membrane and various intracellular structures of absorptive intestinal and renal tubular cells are shown. A comparison is made with preembedding staining results on Concanavalin A-binding site localization in rat liver which shows that problems of penetration common in such a technique are circumvented by the postembedding approach described here. Concanavalin A-binding sites were not only consistently found in nuclear envelope, rough and smooth endoplasmic reticulum, plasma membranes, and collagen fibers, but also in mitochondria, glycogen, ribosomes, and nucleus. These data and those of a previous investigation (Roth J, Cytochem 31:547, 1983) prove the applicability of this cytochemical technique for postembedding localization of glycoconjugates by light and electron microscopy.

Casanova, S; Donini, U; Zini, N; Morelli, R; Zucchelli, P

doi: 10.1177/31.8.6345656pmid: 6345656

Hydroxyethyl-methacrylate (GMA) embedding has recently been proposed for light microscopy studies. In the present investigation extracellular protein antigens were localized on GMA-embedded renal biopsy tissue. Conventionally frozen sections were compared with GMA sections from 55 renal specimens for the detection of extracellular protein antigens. Sections were directly stained with fluorescein- or peroxidase-conjugated antisera against immunoglobulin (Ig) G, IgA, IgM, C3, C1q, and fibrinogen. Results obtained using these two methods showed a 74-89% agreement, depending on the antigen under study. Some discrepancy between GMA and frozen sections was observed in three cases of renal amyloidosis and those cases presenting focal or trace reactions; the differences did not, however, influence the diagnosis. Prerequisites for antigen recovery on GMA sections were a) choice of fixative; b) abrupt dehydration of specimens; and c) treatment of sections with nonspecific protease. The improved localization and the lower background staining obtained led to easy and immediate detection of antigens on GMA sections despite the reduced antigenicity due to the embedding process.

McClung, R E; Wood, J

doi: 10.1177/31.8.6408162pmid: 6408162

Dense bodies containing high amounts of chrome were localized in the perikarya of substantia nigra and dorsal raphe neurons following the cytochemical reaction of endogenous dopamine and serotonin (respectively) with glutaraldehyde-dichromate (GDC). Energy dispersive X-ray analysis of these bodies revealed chrome levels two to four times higher than those recorded from the cytoplasmic background. Pretreatment with paraformaldehyde blocked the GDC reaction within the dense bodies in the substantia nigra (chrome levels similar to background), while the chrome levels in the dense bodies of the raphe neurons remained elevated. This demonstrates that pretreatment with paraformaldehyde allows selective localization of central nervous system serotonin stores by the GDC technique.

Nagle, R B; Clark, V A; McDaniel, K M; Davis, J R

doi: 10.1177/31.8.6190854pmid: 6190854

A comparison of five immunohistochemical methods for the demonstration of keratins in human ovarian neoplasms using affinity-purified polyclonal rabbit antibody was made. The use of indirect immunofluorescence on frozen sections briefly fixed in acetone was found to be the most sensitive method and demonstrated keratin in all 14 primary and 1 metastatic ovarian epithelial neoplasms studied. Protein A-peroxidase, peroxidase--antiperoxidase (PAP), indirect peroxidase, or the avidin--biotinylated peroxidase complex (ABC) methods applied to formalin-fixed tissues were less sensitive and led to false negative results in 9 of 15, 1 of 15, 8 of 15, and 6 of 15 cases, respectively. A single case of dysgerminoma failed to reveal keratin by any method.

Dilley, R; McGeachie, J

doi: 10.1177/31.8.6190855pmid: 6190855

A study was designed to test the suitability of p-phenylenediamine (pPd) as a block stain for light microscope autoradiography. This was done to obviate the conventional method of staining through the emulsion with histological stains after exposure and development. Ten rats were injected with 3H-thymidine and 1 to 3 days later were perfused with glutaraldehyde. Tissue samples were processed by three different schedules: 1) direct embedding in Araldite-Epon; 2) postfixation in OsO4 before embedding; 3) postfixation in OsO4 and staining in 1% pPd before embedding. Autoradiographs of these tissues showed that pPd-treated tissues were well stained with clearly defined cellular detail. The nuclear region was pale-staining, which highlighted the overlying silver grains. There was no evidence of chemography and the nuclear labeling intensity did not differ significantly from the fixed and post-fixed tissues. It was concluded the pPd is a most useful block prestain to use for light microscope autoradiography.

Locke, M; Huie, P

doi: 10.1177/31.8.6190856pmid: 6190856

The Champy-Maillet OsKI reaction has been used upon Golgi complexes to show two kinds of staining. It stains material being processed as it passes along the secretory pathway of the rough endoplasmic reticulum (RER) and Golgi cisternae (GC) up to crystallization in secretory vesicles. It also stains separately the environment within parts of the GC. This GC staining may occur in all compartments (transition vesicles, saccules, condensing vacuoles), but it is characteristically missing from any one of them. The unstained cisternae may be explained if outer saccules are made from either stained or unstained transition vesicles, both of which occur. The presence of empty, unstained transition vesicles is dictated by the surface to volume ratios of microvesicles in relation to saccules. Most transition vesicles must return their membrane to the endoplasmic reticulum, but from time to time it is presumed that they fuse to make a saccule. Saccules, stained and unstained, then mature through the stack. OsKI reactions with tissues and test molecules suggest that in the RER and GC the stain detects labile--S . S--bridges before they lock the tertiary configuration of proteins.

Fleischer, B

doi: 10.1177/31.8.6345657pmid: 6345657

A major role of the Golgi apparatus in liver is the terminal glycosylation of secreted serum proteins and of plasma membrane glycoproteins. Galactosyltransferase is a membrane-bound Golgi enzyme that transfers galactose directly from uridine diphosphogalactose (UDP-Gal) to terminal N-acetylglucosamine groups of N-asparagine-linked glycoproteins during secretion. Sialytransferase then transfers sialic acid from cytidine monophosphosialic acid (CMP-NAN) to the newly added terminal galactose of the glycoprotein. In the cell, the transfer reaction must occur on the lumen side of the Golgi membrane. UDP-Gal is synthesized mainly in the cytoplasm and CMP-NAN is synthesized in the nucleus in liver. An important question for understanding the mechanism is, how do these nucleotide sugars gain access to the transferases? A second question involves uridine diphosphate (UDP), a highly inhibitory product of galactosyltransferase. How is UDP removed from the lumen of the Golgi fast enough to prevent product inhibition of the galactosyltransferase? We have shown that isolated Golgi, although vesiculated, retains its original orientation. The vesicles are oriented with greater than 90% of both galactosyltransferase and sialyl-transferase on the luminal side of the vesicles. Using intact vesicles, we can show that UDP-Gal is taken up via a saturable carrier system present in the Golgi membrane. During galactosylation in vitro, UDP formed in the lumen of Golgi vesicles is rapidly converted to UMP by a nucleoside diphosphatase in the lumen. Uridine monophosphate, which is much less inhibitory to the galactosyltransferase than UDP, is then transported out of the lumen by a second carrier and is broken down further to uridine by 5'-nucleotidase on the cytoplasmic side of the Golgi vesicles. The transport of nucleotides appears unique to the Golgi membranes, since neither rough endoplasmic reticulum nor plasma membrane vesicles from rat liver accumulate these nucleotides.

Oliver, C; Hand, A R

doi: 10.1177/31.8.6134769pmid: 6134769

The distribution of thiamine pyrophosphatase (TPPase) and acid phosphatase (AcPase) has been examined in resting parotid acinar cells as well as during decreased and increased secretory granule production. In resting acinar cells, TPPase activity was restricted to the trans Golgi saccules and AcPase activity was localized in GERL and immature secretory granules. Although secretory granule production is diminished during ethionine intoxication, no significant alteration in the distribution of either TPPase or AcPase was noted. However, marked changes in enzyme localization, especially of TPPase, occurred during accelerated secretory granule production. The alterations were essentially the same for all of the conditions studied (recovery from ethionine treatment, recovery from a protein depletion diet, secretory stimulation with isoproterenol, and postnatal maturation of the parotid gland). During maximal secretory granule production, TPPase activity was localized not only in the trans Golgi saccules, but also in GERL-like cisternae and immature secretory granules. The immature secretory granules were often in continuity with the GERL-like cisternae. At the same time that the TPPase activity was increased, the AcPase activity was frequently diminished. These modulations in enzyme activity provide evidence that GERL is derived from the trans Golgi saccule.

Slot, J W; Geuze, H J

doi: 10.1177/31.8.6863900pmid: 6863900

Using immunogold labeling of ultrathin cryosections, we have studied the localization of secretory, lysosomal, and membrane proteins in the Golgi complexes of several cell types. All proteins were present in the stacks of Golgi cisternae, illustrating that the cisternae comprise a ubiquitous way station for proteins with multiple destinations. The labeling patterns support the concept that peripheral Golgi vesicles represent the main site of secretory protein concentration. Of the membrane proteins studied, the Golgi enzyme galactosyltransferase was confined to the trans-most few cisternae, whereas the receptors for asialoglycoproteins and for polymeric immunoglobulin A occurred in most cisternae, with increasing concentration approaching the trans side. The findings are discussed in relation to a cisternal cis to trans progression of Golgi cisternae and membrane specificity.