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A variety of fixatives, buffers and fixation procedures were compared in rat and squirrel monkey lung in an attempt to preserve optimally both the cytologic details of pulmonary parenchyma as well as the acellular avelolar lining layer. In initial experiments utilizing the fixative of Ito and Karnovsky ('68), an electron-dense deposit was observed on the alveolar surface. Experiments were carried out in an attempt to determine what component of this fixative was responsible for the reaction product observed. In addition,immersion fixation of tissue blocks was compared to the whole lung fixation method of Kikkawa ('70). Kikkawa ('70) achieved excellent preservation of the acellular alveolar lining layer by such a fixation technique. In all lungs examined, whenever a phosphate buffer was utilized with primary aldehyde fixation, an electron-dense precipitate was observed on the luminal surfaces of the type I and II pulmonary epithelial cells. Additional sites of reaction product were pinocytotic vesicles of the type I cells and membranous arrays within the alveolar lumen. Such deposits were never observed when a sodium cacodylate buffer was used. No such granules were observed in areas of lung where the acellular alveolar lining layer had been preserved. The implications of these findings with regard to lung histochemical procedures and the possible relationship of these phosphate buffer-dependent granules to the surfactant system are discussed.
Anat Rec 1975 Mar
PMID:The effects of various fixative-buffer combinations on lung fine structure. 4 21

Adult and half grown healthy male and female cats were used in this study. The white and gray matter of dorsolateral region in the upper lumbar levels of the spinal cord were examined by electron microscopy after fixation by aldehyde perfusion and commonly used methods of embedding, sectioning and staining. The report is limited to description and illustration of specialized junctions of astrocytes. In addition to previously described astrocyte-astrocyte gap junctions, astrocytes are connected by gap junctions to oligodendroglia cells and to neurons. Astrocytes also are connected with each other and with neurons by juctions characterized by wide (250 A) gaps containing opaque gap material and by dense material adhering to the inner surfaces of the plasma membranes. The results suggest a morphological basis for adhesion and intercommunication between all adult derivatives of the embryonic neural tube.
Anat Rec 1975 Jun
PMID:Specialized contacts of astrocytes with astrocytes and with other cell types in the spinal cord of the cat. 5 Jul 52

A reliable and uniform vascular perfusion fixation method for the testis has been developed by using an initial washout solution containing a vasodilator and an anticoagulant. This is followed by a brief fixation with a sodium phosphate buffered formaldehyde-glutaraldehyde solution of conventional strenght, and then a second more concentrated aldehyde fixative solution containing picric acid. The method takes into account some of the unique features of the vascular supply of the male genital tract for its favorable perfusion and fixation. The advantages of this method are: (1) consistently favorable preservation of the testis; (2) simple and inexpensive apparatus; and (3) stable and relatively innocuous stock solutions.
Anat Rec 1977 Jul
PMID:An improved perfusion fixation method for the testis. 33 10

White adipose tissue was obtained from the mesentery, epididymis, omentum and subcutis of rats which were fed, fasted or fasted and then refed. Tissue samples were prepared using the glyoxylic acid method to detect adrenergic nerves by fluorescence histochemistry. Other tissue samples were fixed with an aldehyde solution containing sodium molybdate which is specific for catecholamine granules in nerve terminals. Thin and serial thick sections (0.25-0.5 micron) were viewed with a conventional electron microscope and with the high voltage electron microscope. With fluorescence microscopy it was found that most of the blood vessels except veins and venules were richly innervated. The most extensive branching of nerves down to the capillary level was found in the mesentery and epididymal fat of fasted-refed rats. Relatively few adipocytes appeared to be innervated. With electron microscopy, nerve terminals were found distributed with most blood vessels including capillaries, and with some adipocytes. Only 2-3% of all dipocytes were innervated by adrenergic nerves. It is suggested that in the adipose tissue sites studied the major adrenergic innervation is mainly for the supply of blood vessels.
Anat Rec 1978 Jul
PMID:Morphological studies on the adrenergic innervation of white adipose tissue. 67 91

Osteopenia is a recognized complication of diabetes mellitus in humans and experimental animals. We recently found that tetracyclines prevent osteopenia in the streptozotocin-induced diabetic rat and that this effect was associated with a restoration of defective osteoblast morphology (Golub et al., 1990). The present study extends these initial ultrastructural observations by assessing osteoblast function in the untreated and tetracycline-treated diabetic rats. After a 3-week protocol, non-diabetic control and diabetic rats, including those orally administered a tetracycline, minocycline (MC), or a non-antimicrobial tetracycline analog (CMT), were perfusion-fixed with an aldehyde mixture; the humeri were dissected and processed for ultracytochemical localization of alkaline phosphatase (ALPase) and Ca-ATPase activities. Some rats from each experimental group received an intravenous injection of 3H-proline as a radioprecursor of procollagen, and the humeri were processed for light microscopic autoradiography. In addition, the osteoid volume in each experimental group was quantitatively examined by morphometric analysis of electron micrographs. During the diabetic state, active cuboidal osteoblasts in the endosteum of control rats were replaced by flattened bone-lining cells that contained few cytoplasmic organelles for protein synthesis (Golgi-RER system), and active transport (mitochondria). Treating diabetic rats with MC, and even more so with CMT, appeared to "restore" osteoblast structure. During diabetes, bone-lining cells incorporated little 3H-proline or secreted little labeled protein and produced only a very thin osteoid layer. Tetracycline administration to the diabetics increased both the incorporation of 3H-proline by osteoblasts and their secretion of labeled protein toward the osteoid matrix, in a pattern similar to that seen in the non-diabetic controls.(ABSTRACT TRUNCATED AT 250 WORDS)
Anat Rec 1991 Sep
PMID:Tetracycline administration restores osteoblast structure and function during experimental diabetes. 183 18

Muscle spindles in the tenuissimus muscle of mature golden Syrian hamsters were examined by conventional and high-resolution scanning electron microscopy (HRSEM). For conventional SEM, entire muscles were first fixed in 2.5% buffered glutaraldehyde. Spindles were then isolated with a dissecting microscope under darkfield illumination and postfixed in 1.0% OsO4. Some spindles were treated with 8 N HCl at 60 degrees C to clearly expose intrafusal fiber surfaces once the outer capsular sheath was mechanically disrupted. Preparation for HRSEM included aldehyde/osmium fixation and freeze-cleavage in liquid N2. The cytosol and certain cellular elements were also selectively extracted by immersion in 0.1% OsO4 for varying time intervals. In these preparations, the capsular sleeve showed a multilayered pattern of vesicle-laden cells with variant surface topography in different regions, including filopodia and small bristle-like surface-projections. An interlacing three-dimensional network of collagen fibrils intervened between the capsular lamellae. Within the spindles, sensory and fusimotor nerve endings closely adhered to the outer surfaces of intrafusal fibers. Sensory nerve terminals were enveloped by a prominent external lamina, and those that were cleaved open contained a plethora of elongated mitochondria that ran parallel with the longitudinal axis, along with vesicles, axoplasmic filaments, and lysosomes. Multiple adhesion sites between the sensory nerve membrane and the underlying sarcolemma of the intrafusal fiber were also observed in select regions. Fusimotor nerve endings were covered externally by processes of Schwann cells and their axoplasm was filled with a multitude of cellular organelles and synaptic vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)
Anat Rec 1991 Jun
PMID:Muscle spindle ultrastructure revealed by conventional and high-resolution scanning electron microscopy. 186 95

The distribution and cellular localization of the glycoprotein laminin were investigated by light and electron microscopic immunocytochemistry in the adult murine pituitary gland. Immunoblots confirmed that laminin was the only protein in the pituitary gland of the adult male mouse to react with antilaminin serum. Laminin immunoreactivity was demonstrated at the light microscopic level simultaneously with that of beta-follicle stimulating hormone (beta-FSH) and beta-luteinizing hormone (beta-LH). In addition to its distribution is basal laminae, laminin immunoreactivity was coincidently expressed in gonadotrophs with the immunoreactivities of beta-FSH and beta-LH. Electron microscopic immunocytochemistry was employed on aldehyde-fixed sections embedded in L.R. White. Sites of binding of primary antisera to laminin were identified with affinity-purified secondary antisera directly coupled to 20 nm particles of colloidal gold. Three antisera recognizing laminin were compared and found to result in an identical pattern of immunoreactivity. Laminin was found extracellularly only in formed basal laminae in all three lobes of the pituitary and was not found in extracellular matrices of connective tissue. Laminin immunoreactivity was also found intracellularly in gonadotrophs but in none of the other endocrine or non-endocrine cells of the anterior lobe. Within gonadotrophs, only secretory granules were labeled. The majority, but not all, secretory granules were labeled in each of the gonadotrophs examined, and the proportion of granules labeled with laminin could not be increased by doubling the concentration of anti-laminin serum. Laminin immunoreactivity segregated with the subset of secretory granules containing beta-FSH. In contrast, laminin immunoreactivity was absent in the smaller subset of secretory granules that contain serotonin.(ABSTRACT TRUNCATED AT 250 WORDS)
Anat Rec 1990 Apr
PMID:Distribution of laminin in the murine pituitary. 210 52

The migration of intestinal intervillous epithelial cells labeled in the fetus was followed in neonatal mice. At 17 days of gestation, a first group of pregnant mice received three intraperitoneal injections of 3H-thymidine (150 microCi/injection) administered at 30 min intervals. Two mothers were sacrificed 3 hours after the first injection. Mice from different litters were also sacrificed on days 0, 2, 4, 8, 12, 14, and 16 after birth. A second group of pregnant mice was injected at 18 1/2 days of gestation and offspring were sacrificed on days 6, 8, 10, 12, 14, and 16 after birth. Segments of duodenum and ileum were fixed in glutaraldehyde, postfixed in osmium tetroxide, dehydrated, and embedded in Epon. Sections were stained with aldehyde fuchsin and processed for radioautography. By following the leading front and trailing edge of labeled cells in the longest villi of the duodenum and ileum, we observed that 1) extrusion zones become active immediately after birth and 2) the longest villi do not elongate until 10 days after birth in the duodenum and 14 days in the ileum, that is, when all labeled epithelial cells originally present in the fetus have been extruded. Moreover, by measuring the distance between the internal limit of the inner circular layer of smooth muscle and the intervillous epithelium at 17 days of gestation (12.95 +/- 1.18 microns) or the bottom of the crypts at day 3 (14.81 +/- 0.91 microns), we propose that crypts do not develop as downgrowths: rather the intervillous epithelium is reshaped and the crypt-villus junction moves upward, away from the muscularis externa.(ABSTRACT TRUNCATED AT 250 WORDS)
Anat Rec 1990 Jun
PMID:Migration of fetal intestinal intervillous cells in neonatal mice. 235 8

Our recent observation that the basement membranes of brain microvessels do not stain with the cationic dye ruthenium red has raised the question of whether the basement membranes of this and other vascular beds functioning as barriers between blood and neural tissues are deficient in the polyanionic macromolecules, such as glycosaminoglycans, which are responsible for the ruthenium red staining of other vascular basement membranes. We therefore attempted to produce staining in the only barrier-type microvascular basement membrane known to contain heparan sulfate. Bovine retinas were fixed by immersion in aldehyde fixatives containing ruthenium red, buffered with either 10 mM or 100 mM sodium cacodylate. We found discrete, electron-dense deposits of ruthenium red in vascular basement membranes, quite similar to those seen in vascular basement membranes of nonneural tissues after exposure to ruthenium red. These deposits were more distinct and more frequent in tissue exposed to ruthenium red-aldehyde solutions buffered with 10 mM cacodylate. They were not seen if ruthenium red was omitted from the fixative. The results demonstrate that anionic macromolecules in basement membranes of barrier-type microvessels can be stained with cationic dyes, and suggest that the failure of brain microvessels to stain with ruthenium red may be the result of a relative or total lack of polyanion in this basement membrane, or of other unique properties.
Anat Rec 1987 Dec
PMID:Ultrastructural studies of bovine retinal microvascular basement membranes with the cationic dye ruthenium red. 245 89

Ectomesenchyme derived from cardiac neural crest is critical to aorticopulmonary septation in the heart. However, any unique contribution of the cardiac ectomesenchyme to the extracellular matrix of the conotruncus has not been demonstrated previously. In this study the chronology and topography of soluble tropoelastin (STE) and the aldehyde-rich protein (ARP) of the elastic connective tissues have been examined in the chick embryo, stages 21-38, and in the quail-chick chimera, stages 24-35 (quail neural fold grafted onto a chick embryo). STE was located with immunofluorescence histochemistry, and ARP with Schiff's reagent. With these procedures prevenient sites of elastin synthesis are observed readily. The results show that the myocardium proper appears to have a role in the instigation of elastogenesis and in elastic fiber orientation; that the mesenchymal cells whose matrix contains elastic fibers are ectomesenchymal, of neural crest origin; and that elastin is deployed in an orderly proximal-distal sequence. It is hypothesized that elastogenesis is a critical event in aorticopulmonary septation.
Anat Rec 1988 Aug
PMID:Origin and propagation of elastogenesis in the developing cardiovascular system. 305 14


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