Journal of Histochemistry & Cytochemistry

Bucana, C; Saiki, I; Nayar, R

doi: 10.1177/34.9.2426339pmid: 2426339

Hydroethidine, a reduced form of ethidium bromide, was used as a vital dye in fluorescence assays that allowed visual and semiquantitative monitoring of dye uptake and accumulation by fluorescence microscopy, flow cytometry, image analysis, and microfluorimetry. The excitation and emission filters were chosen to detect hydroethidine and exclude ethidium. Microscopically, there were differences in fluorescence intensities and fluorescence patterns among various tumor cell lines. The fluorescence pattern varied from homogeneous blue in the cytoplasm to blue plus brilliant packets of bluish-white distributed in the cytoplasm. Nuclear staining varied from brown to reddish orange fluorescence. These differences were confirmed by flow cytometry and image analysis. A preliminary survey of various tumors indicated that uptake and accumulation of hydroethidine were dependent on concentration of the dye, duration of cell exposure to the dye, and metabolic state of the cells. Microfluorimetry made possible monitoring of 96 samples in a microculture plate in 30 seconds; thus, this method allows large numbers of samples to be read, with a tremendous savings in time and reagents. The results obtained from the different techniques used were corroborative; therefore, any one of the above techniques may be used in an assay.

Vaillant, C R; Lund, P K

doi: 10.1177/34.9.3755450pmid: 3755450

Recently, a putative hormone, glucagon-like peptide I (GLP I), has been identified in the predicted sequences of the precursors to pancreatic glucagon in human, rat, hamster, and ox. The distribution of GLP I immunoreactivity in canine and feline pancreas and gastrointestinal tract was examined immunohistochemically and was compared with that of two other antigenic determinants of pancreatic pro-glucagon, i.e., glucagon and the NH2 terminus of glicentin. All three determinants occurred in the same population of islet cells in normal pancreas and in pancreas consisting predominantly of islet tissue from dogs with canine pancreatic acinar atrophy. Northern blot analysis of mRNA from the latter tissue, using a rat pre-pro-glucagon complementary DNA probe, revealed a single mRNA species similar in size to the pre-pro-glucagon mRNA detected in fetal rat pancreas. The three antigenic determinants of pancreatic pro-glucagon were co-localized also in intestinal L-cells and in canine gastric A-cells. Canine and feline pancreatic pro-glucagons therefore resemble those identified in other mammals and may also occur in gastrointestinal endocrine cells. Although there is evidence that the GLP I sequence is not liberated from pancreatic pro-glucagon, our results raise the possibility that this putative hormone may be a cleavage product of pro-glucagon in the gastrointestinal tract.

Linner, J G; Livesey, S A; Harrison, D S; Steiner, A L

doi: 10.1177/34.9.2426340pmid: 2426340

An apparatus has been produced that can remove amorphous phase tissue water via molecular distillation without devitrification or rehydration. This method represents a fundamental advance in tissue preparation, making possible for the first time ultrastructural localization of soluble molecular entities without the problems of alteration, re-distribution, and loss which have plagued conventional techniques. Fresh slices of rat brain, liver, or kidney, and monkey retinal tissue were cryofixed by bounce-free, metal mirror cooling on copper bars immersed in liquid nitrogen (LN2). Tissue transferred under LN2 was then placed in a precooled copper specimen block, which was subsequently lowered into a LN2-cooled stainless steel chamber. After rough pumping at 1 X 10(-3) mbar with a mechanical pump to remove LN2, the chamber was evacuated with a cryopump or turbomolecular pump to achieve a hydrocarbon-free, ultra-high vacuum of 1 X 10(-8) mbar. Equilibrium temperature in the chamber before the drying cycle was -192 degrees C. The copper specimen block was equipped with a thermocouple and a programmable feedback-controlled heating circuit. Tissue was dried by increasing the specimen block temperature 1 degree C/hr during the critical drying phase while monitoring the rate of water removal with a partial pressure analyzer. Results obtained indicate that drying is complete below the devitrification temperature of amorphous phase tissue water. Dried tissue was fixed with osmium tetroxide vapor, vacuum-embedded in a low-viscosity epoxy resin, sectioned, stained, and viewed with the electron microscope. Processed tissue exhibits excellent morphological preservation without the use of pre-fixation or cryoprotective agents. Thin sections of this tissue are excellent for immunocytochemical staining and electron microprobe analysis.

Walker, S R; Williams, M C; Benson, B

doi: 10.1177/34.9.2426341pmid: 2426341

The adsorptive properties of phospholipids of pulmonary surfactant are markedly influenced by the presence of three related proteins (26-38 KD, reduced) found in purified surfactant. Whether these proteins are pre-assembled with lipids before secretion is uncertain but would be expected for a lipoprotein secretion. We performed indirect immunocytochemistry on frozen thin sections of rat lung to identify cells and intracellular organelles that contain these proteins. The three proteins, purified from lavaged surfactant, were used to generate antisera in rabbits. Immunoblotting of rat surfactant showed that the IgG reacted with the three proteins and a 55-60 KD band which may be a polymer of the lower MW species. Specific gold labeling occurred over alveolar type II cells, bronchiolar Clara cells, alveolar macrophages, and tubular myelin. In type II cells labeling occurred in synthetic organelles and lamellar bodies, which contain surfactant lipids. Lamellar body labeling was increased fivefold by pre-treating tissue sections with a detergent. Multivesicular bodies and some small apical vesicles in type II cells were also labeled. Secondary lysosomes of alveolar macrophages were immunoreactive. Labeling in Clara cells exceeded that of type II cells, with prominent labeling in secretory granules, Golgi apparatus, and endoplasmic reticulum. These observations clarify the organelles and pathways utilized in the elaboration of surfactant. After synthesis, the proteins move, probably via multivesicular bodies, to lamellar bodies. Both lipids and proteins are present in tubular myelin. Immunologically identical or closely similar proteins are synthesized by Clara cells and secreted from granules which appear not to contain lipid. The role of these proteins in bronchiolar function is unknown.

Hunziker, E B; Graber, W

doi: 10.1177/34.9.2426342pmid: 2426342

Small tissue blocks of native rat growth plate cartilage were incubated for short periods in one of several generally used isotonic buffer salt solutions or commercial tissue-culture media. The total percentage (approximately 12) of [35S]-labeled proteoglycans (PG) extracted from cartilage matrix under these conditions was not significantly influenced by either the chemical composition of the medium or the presence of a protease inhibitor. Morphological examination of incubated tissue after fixation in the presence of ruthenium hexamine trichloride (RHT) (included to preserve PG in situ) revealed, however, that the PG staining profiles across cartilage matrix varied with the composition of the incubation medium used. The various susceptibilities exhibited by PG within the different matrix compartments to selective extraction was estimated semi-quantitatively. The observed effects may prove useful in extracting these molecules differentially from cartilage matrix compartments.

Salido, E C; Barajas, L; Lechago, J; Laborde, N P; Fisher, D A

doi: 10.1177/34.9.2426343pmid: 2426343

Epidermal growth factor (EGF) was originally isolated from mouse submandibular glands (SMG). However, SMG removal failed to lower circulating EGF, and large amounts of EGF have been found in mouse urine. In addition, the presence of pre-pro-EGF mRNA in mouse kidney has recently been reported by others. Kidneys may therefore represent an alternate source of EGF. In the present study, we investigated the immunocytochemical localization of EGF in mouse kidney. Male and female adult Swiss Webster mice were fixed by perfusion with 4% paraformaldehyde or Zamboni's fixative, the kidneys were frozen, and serial sections were obtained. Rabbit EGF antiserum was used for the primary incubation and the avidin-biotin complex immunoperoxidase procedure was utilized for immunostaining. EGF was immunolocalized in the apical portion of the cells lining the thick ascending limb of Henle (TALH) and the distal convoluted tubule (DCT). The macula densa, in contrast, lacked EGF immunoreactivity. No sex differences were observed in the distribution pattern or intensity of immunostaining. Infusion of EGF into sheep renal artery has been reported to induce changes in urine flow and ionic composition. Immunolocalization of EGF in the TALH and DCT documented here supports a regulatory role for EGF in the function of the mouse distal nephron.

Derenzini, M; Farabegoli, F; Marinozzi, V

doi: 10.1177/34.9.2426344pmid: 2426344

The periodic acid-thiocarbohydrazide or thiosemicarbazide-OsO4 method (Seligman AM, Hanker JS, Wasserkrug H, Katzoff L: J Histochem Cytochem 13:629, 1965) has been modified in order to obtain a periodic acid-Schiff (PAS)-like reaction for electron microscopy capable of visualizing structures at the molecular level in situ. Thiocarbohydrazide (TCH) and thiosemicarbazide (TSC) have been used dissolved in distilled water and bubbled with SO2. Treatment of previously oxidized thin sections with TCH (SO2) or TSC (SO2), followed by osmification, resulted in selective and very good staining of all the PAS-positive structures examined: glycogen, intestinal mucopolysaccharides, plasma membrane glycoproteins, basement membranes, Golgi apparatus, and collagen. The staining reaction was highly specific when TSC was used on thin sections from paraformaldehyde-fixed samples. The non-particulate end-reaction product made possible visualization of a periodic distribution of sugar residues in the 64-nm unit of collagen and the structural organization of the PAS-positive glycoconjugate components in the glomerular basement membrane.

Weinman, S; Ores-Carton, C; Rainteau, D; Puszkin, S

doi: 10.1177/34.9.2426345pmid: 2426345

Using the Lowicryl K4M embedding technique, together with indirect immunoferritin or immunogold labeling on ultra-thin sections, tubulin, calmodulin and phospholipase A2 were distinctly localized in ejaculated bull spermatozoa. Calmodulin was concentrated on the plasma membrane, nucleus, post-acrosomal substance, and, in lesser amounts, between coarse fibers and axonemal microtubules of the flagellum. Phospholipase A2 was distributed evenly along the plasma membrane, nucleus, acrosome, post-acrosomal substance, and in the flagellum, on mitochondria, fibrous sheath, coarse fibers, between coarse fibers and axonemal microtubules. Antibodies to tubulin labeled only axonemal microtubules, including the central pair of microtubules. Patterns of tubulin labeling were identical when ferritin granule- or gold particle-conjugated antibodies were tested. In agreement with our previous biochemical studies demonstrating calmodulin binding to phospholipase A2, concomitant with enhancement of phospholipase A2 activity (Arch Biochem Biophys 241:413, 1985), the overlapping distribution of calmodulin and phospholipase A2 in several parts of the sperm suggests that these proteins may play a concerted role in male gamete function in preparation for or during fertilization. The distinct distribution of tubulin along flagellum microtubules indicates their special function in sperm mobility.

Weinman, S; Ores-Carton, C; Escaig, F; Feinberg, J; Puszkin, S

doi: 10.1177/34.9.3734420pmid: 3734420

Affinity-purified monospecific antibodies and indirect immunogold and immunoferritin labeling on ultra-thin sections of low-temperature Lowicryl K4M-embedded samples were used to study the redistribution of calmodulin in ram spermatids and epididymal spermatozoa at the electron microscopic level. Calmodulin appeared as an integral component of well-defined structures or organelles of these cells. In young spermatids, calmodulin was localized in the nucleus, cytoplasm, and developing acrosome. During spermatogenesis and epididymal maturation, calmodulin left the acrosome to reach the perinuclear substance and finally became concentrated in the post-acrosomal area of the head, although some calmodulin remained associated with the tip of the acrosome. Such a redistribution is consistent with the preferential location of Ca2+ in the post-acrosomal cytoplasm of ejaculated spermatozoa. Calmodulin was also observed in the flagellum associated with the plasma membrane and with the motility apparatus, between coarse fibers and axonemal microtubules. These changes in calmodulin distribution may account for the Ca2+-dependent regulation of spermatogenesis and sperm maturation. Calmodulin therefore appears to be a pleiotropic regulator of male gamete development and functions.

Yorde, D E; Kalkhoff, R K

doi: 10.1177/34.9.3525667pmid: 3525667

We have developed a computerized system for quantitative morphometric analysis of the number and position of secretory granules and organelles in pancreatic islet beta cells following tolbutamide treatment. Data from animals injected with tolbutamide for 1, 2, and 3 days were compared to tissues obtained from untreated control animals. Pancreatic islets removed by a collagenase technique were perfused with an appropriate medium to restore a basal state. After fixation and embedding, thick sections of beta cells were viewed by electron microscopy. Morphometric studies of randomly selected or serially cut cells were performed with computer programs for digitization, quantify, rotational, and perspective display. Tolbutamide treatment resulted in graded granule depletion which was maximal at 72 hr relative to control animals. Reduced granule density was associated with significant reduction in total cell area or cytoplasmic area, but was without effect on nuclear size. Since granule depletion improved visualization of subcellular structures, this will enable us to pursue studies of exocytosis under a variety of physiological conditions.