Journal of Histochemistry & Cytochemistry

Schmidt, H H; Gagne, G D; Nakane, M; Pollock, J S; Miller, M F; Murad, F

doi: 10.1177/40.10.1382087pmid: 1382087

Nitric oxide synthases (NOS Types I-III) generate nitric oxide (NO), which in turn activates soluble guanylyl cyclase (GC-S). The distribution of this NO-mediated (nitrinergic) signal transduction pathway in the body is unclear. A polyclonal monospecific antibody to rat cerebellum NOS-I and a monoclonal antibody to rat lung GC-S were employed to localize the protein components of this pathway in different rat organs and tissues. We confirmed the localization of NOS-I in neurons of the central and peripheral nervous system, where NO may regulate cerebral blood flow and mediate long-term potentiation. GC-S was located in NOS-negative neurons, indicating that NO acts as an intercellular signal molecule or neurotransmitter. However, NOS-I was not confined to neurons but was widely distributed over several non-neural cell types and tissues. These included glia cells, macula densa of kidney, epithelial cells of lung, uterus, and stomach, and islets of Langerhans. Our findings suggest that NOS-I is the most widely distributed isoform of NOS and, in addition to its neural functions, regulates secretion and non-vascular smooth muscle function. With the exception of bone tissue, NADPH-diaphorase (NADPH-d) activity was generally co-localized with NOS-I immunoreactivity in both neural and non-neural cells, and is a suitable histochemical marker for NOS-I but not a selective neuronal marker.

Adams, J C

doi: 10.1177/40.10.1527370pmid: 1527370

A procedure is described for intensifying histochemical reactions by amplification of biotinylated sites. This is achieved by deposition of biotinylated tyramine on the tissue through the enzymatic action of horseradish peroxidase (HRP). The amplified biotin sites are subsequently visualized by binding them to avidin, to which a marker is attached. This amplification greatly increases the sensitivity of staining procedures that employ HRP (and/or biotin) in tissue. For neuroanatomical pathway tracing methods, the procedure greatly increases the detectability of the injected tracer. For lectin histochemistry and immunohistochemistry, the amplification requires that the lectin or primary antibody be greatly diluted. This dilution results in less background staining and yet strong signals are produced even when very dilute reagents are used. Alternatively, the amplification permits much shorter incubations in primary antibodies when dilutions are used that would ordinarily be used with conventional bridge techniques. The procedure is also useful for amplifying very weak signals, such as those of immunoreactions in glutaraldehyde-fixed tissue. The amplification procedure, together with the availability of avidin probes labeled with fluorochromes, colloidal gold, or enzyme systems other than HRP, provides a means of greatly increasing the versatility of a variety of histochemical reactions, including those for detecting in situ hybridization probes, in addition to increasing the sensitivity of the reactions.

Marshak, D W

doi: 10.1177/40.10.1356122pmid: 1356122

The goal of this study was to develop an alternative to silver intensification for visualizing small colloidal gold particles by light and electron microscopy. The isolated goldfish retina was labeled with rabbit antiserum to tyrosine hydroxylase and 1-nm colloidal gold-conjugated goat anti-rabbit IgG. The gold particles were enlarged by toning with gold chloride, followed by reduction in oxalic acid. Dopaminergic interplexiform cells were clearly visible by light microscopy and, in lightly-fixed material treated with detergent, they were labeled in their entirety. Labeling was qualitatively similar, although less extensive, in material fixed and processed for electron microscopy. The labeled processes were apparent in ultra-thin sections viewed at low magnification, but the gold-toned particles were not so large that they obscured subcellular structures. The procedure apparently had no deleterious effects on the tissue, since the ultrastructural preservation was comparable to that seen with other pre-embedding immunolabeling methods. The technique was simple, reliable and, since the gold solutions were so dilute, relatively inexpensive.

Stahlman, M T; Gray, M E; Whitsett, J A

doi: 10.1177/40.10.1527371pmid: 1527371

The distribution of immunoreactive surfactant-associated protein B (IR-SP-B) was studied immunohistochemically in 120 subjects from 10 weeks of gestation to 7 postnatal months with a polyclonal antibody against human SP-B. Electron microscopy (EM) was done in 72 subjects to document the presence of Type II cells containing lamellar bodies. Fetuses of less than 18 weeks' gestation showed no immunostaining. Beginning at 18 weeks, non-mucous cells of tracheal glands immunostained in a few instances. Fetuses of 19 through 23 weeks showed progressive immunostaining of cells lining terminal airways. Infants 26-40 weeks who died with or without pulmonary pathology showed immunostaining of Type II cells and bronchioloalveolar (BA) portal cells of the respiratory bronchioles. In infants with hyaline membrane disease (HMD) who died less than 12 days after birth, occasional tracheal gland cells, BA portal cells, and mature and relining Type II cells immunostained. In bronchopulmonary dysplasia (BPD), BA portal cells, relining Type II cells, macrophages, and luminal material immunostained. Occasional tracheal and bronchial gland cells and Clara cells immunostained. The appearance of IR-SP-B at mid-gestation correlated with differentiation of Type II cells. There was good correlation of immunostaining with the presence of lamellar bodies on EM. Accelerated maturation of the lung was often associated with premature rupture of membranes (PROM).

Christensen, E I; Gliemann, J; Moestrup, S K

doi: 10.1177/40.10.1382088pmid: 1382088

gp330, a large glycoprotein located in renal proximal tubules, has sequence similarities with the low-density lipoprotein receptor and the alpha 2-macroglobulin receptor/low-density lipoprotein receptor-related protein. The 40 KD human alpha 2-macroglobulin receptor-associated protein is a newly discovered heparin binding protein homologous to a major rat Heymann nephritis factor and exhibiting high affinity binding to the alpha 2-macroglobulin receptor. The present study shows by ligand blotting, light and electron microscopic autoradiography, and cytochemistry that gp330 located in coated apical membrane regions of the rat proximal tubule strongly binds the 40 KD protein. Furthermore, 45Ca2+ blotting experiments disclosed gp330 as a quantitatively important Ca2+ binding protein in renal cortex. Binding of 125I-labeled 40 KD protein to electroblotted gp330 and to coated apical membrane regions in sections of renal proximal tubules was abolished by excess unlabeled 40 KD protein, heparin, and EDTA. The endocytic properties of gp330 were investigated by in vivo microperfusion of rat proximal tubules. After 6 min, 125I-labeled 40 KD protein was mainly found in endocytic vacuoles and later accumulated in lysosomes. These data demonstrate that gp330 is a Ca2+ binding receptor for endocytosis of protein and is functionally related to the alpha 2-macroglobulin receptor/low-density lipoprotein receptor-related protein. Furthermore, our results demonstrate the usefulness of semi-thin and ultra-thin cryosections in studies of ligand binding and subcellular localization of receptors with autoradiographic techniques.

Haller, E M; Shelley, S A; Montgomery, M R; Balis, J U

doi: 10.1177/40.10.1527372pmid: 1527372

Using immunogold labeling of fixed, cryosubstituted tissue sections, we compared the distribution of lysozyme, an oxidant-sensitive lamellar body protein, with that of surfactant protein A (SP-A) in rat Type II cells, extracellular surfactant forms, and alveolar macrophages. Morphometric analysis of gold particle distribution revealed that lysozyme and SP-A were present throughout the secretory and endosomal pathways of Type II cells, with prominent localization of lysozyme in the peripheral compartment of lamellar bodies. All extracellular surfactant forms were labeled for both proteins with preferential labeling of tubular myelin and unilamellar vesicles. Labeling of tubular myelin for SP-A was striking when compared with that of lamellar bodies and other extracellular surfactant forms. Lamellar body-like forms and multilamellar structures were uniformly labeled for lysozyme, suggesting that this protein is rapidly redistributed within these forms after secretion of lysozyme-laden lamellar bodies. By contrast, increased labeling for SP-A was observed over peripheral membranes of lamellar body-like forms and multilamellar structures, apparently reflecting progressive SP-A enrichment of these membranes during tubular myelin formation. The results indicate that lysozyme is an integral component of the lamellar body peripheral compartment and secreted surfactant membranes, and support the concept that lysozyme may participate in the structural organization of lung surfactant.

Seddiki, T; Delpal, S; Ollivier-Bousquet, M

doi: 10.1177/40.10.1527373pmid: 1527373

To study the transcytosis and segregation of ligand in the mammary epithelial cell, endocytosis and intracellular transit of human blood transferrin were followed in lactating rabbit mammary epithelial cells. Human transferrin labeled with biotin added to an incubation medium was bound to the basal membrane of mammary epithelial cells and carried across the cell to the lumen of the acini within 5-60 min. At the same time, biotinylated human transferrin accumulated at the apex of the cell. After incubation with human transferrin labeled with colloidal gold, label was detected inside endosome-like structures, vesicles and saccules of the Golgi apparatus, and inside the lumen within 2-5 min. A significant label accumulated at the apex of the cell after 30-60 min. Biotin labeling did not modify the time of transit of human transferrin, as attested by comparison with the time of transit of native transferrin. Human transferrin was never detected inside vesicles containing casein micelles. In contrast, rabbit milk transferrin was immunocytochemically detected inside vesicles containing casein micelles. These results indicate that transcytosis of human transferrin follows a pathway different from vesicles that carry casein micelles.

Kirkeby, S; Bøg-Hansen, T C; Moe, D

doi: 10.1177/40.10.1382089pmid: 1382089

We compared the localizations of lectin binding and activity for myosin ATPase and succinic dehydrogenase in sections of the gracilis, soleus, and masseter muscles from 10- and 60-day-old rats. In the 60-day-old rats, incubation of the muscle sections with the lectins ConA, GS-II, HPA, and jacalin gave rise to a mosaic staining pattern, especially in the gracilis muscle, in which the same fibers were strongly stained for ConA, GS-II, and HPA, whereas the staining with jacalin in these fibers was weak, and vice versa. There was no correspondence in the staining patterns for the enzymes and the lectins. In the masseter muscle only GS-II gave rise to distinct differences in the staining intensity between muscle fibers. In 10-day-old rats all fibers in the muscles were moderately stained with ConA, HPA, and jacalin, whereas a chessboard staining pattern could be observed after incubation with GS-II. In an extract of hindleg muscle from 60-day-old rats there was strong affinity for ConA and HPA and weak affinity for GS-II and jacalin, as shown by dot-blotting. After electrophoresis and blotting to nitrocellulose membranes, three muscle protein bands with apparent molecular weights of 100,000, 90,000, and 43,000 showed affinity for ConA, HPA, and GS-II, whereas no bands were jacalin positive. The complex lectin staining pattern in skeletal muscle might be related to development, specialization, and function of the muscles.

Kivelä, T; Fuchs, U; Tarkkanen, A

doi: 10.1177/40.10.1382090pmid: 1382090

The cytoskeleton of epithelial and muscle cells of the human iris and ciliary body was analyzed by immunohistochemistry in three morphologically normal formalin-fixed, paraffin-embedded eyes and in 34 eyes containing a uveal melanoma. Both layers of the iris epithelium reacted with monoclonal antibodies (MAb) V9 and Vim 3B4 to vimentin, whereas the ciliary epithelia additionally reacted with MAb CAM 5.2, CK5, KS-B17.2, and CY-90, recognizing cytokeratins 8 and 18. The same cytokeratin MAb labeled the retinal pigment epithelium, which lacked vimentin. The muscle portion of the anterior iris epithelium, which forms the dilator muscle, as well as the sphincter and ciliary muscles, reacted with MAb DE-U-10 to desmin and 1A4 to alpha-smooth muscle actin. The dilator and ciliary muscles also reacted with V9 and Vim 3B4 to vimentin, and some dilator fibers were weakly immunopositive for cytokeratin 8 and 18 with CY-90 and CAM 5.2. The antigenic profile of iris and ciliary epithelia infiltrated by melanoma cells remained unchanged. The intraocular epithelia, which are developmentally related but differ in function, and the intraocular muscles, which differ in origin but are functionally related, have distinct cytoskeletal profiles and may provide insights into the functional significance of intermediate filament expression.

Lacasse, J; Martin, L H

doi: 10.1177/40.10.1382091pmid: 1382091

Cluster of differentiation 1 (CD1) antigens are a family of non-MHC but Class I-like molecules that have been identified in humans and rodents. Although their function(s) remains unknown, it has been proposed that CD1 may present antigens to specific subsets of peripheral T-cells. We now provide evidence in support of this hypothesis through the demonstration by in situ hybridization that Paneth cells of the mouse intestine express CD1 mRNA. These cells are thought to be involved in the immunological regulation of intestinal flora and could accomplish this task through interactions with intestinal intraepithelial lymphocytes. The expression and localization of CD1 mRNA was confirmed by both autoradiographic and non-isotopic techniques. The relevance of these results to CD1 function as well as to Paneth cell biology is discussed.