Journal of Histochemistry & Cytochemistry

Laurie, G W; Leblond, C P

doi: 10.1177/30.10.6290565pmid: 6290565

The parietal layer of the rat yolk sac includes a 5 microliter thick sheet known as Reichert's membrane that exhibits properties of basement membranes. Its inner side is lined by a single layer of loosely distributed cells referred to as endodermal cells. Both Reichert's membrane and endodermal cells were examined at 13-14 days' gestation with emphasis on the ultrastructure of the Golgi apparatus, the identification of its component parts by specific phosphatase activities, and its possible role in the cells' secretory process. Reichert's membrane is composed of a series of stacked layers similar to basal laminae and composed of a network of fibrils with a diameter of 2-8 nm along which dots are located at irregular intervals. The endodermal cells contain the usual organelles, including interconnected rough endoplasmic reticulum (rER) cisternae and a prominent Golgi apparatus. With the help of phosphatase reactions, the stacks of Golgi saccules were divided into a) "phosphatase-free" saccules, the first ones on the cis or forming side, b) one or two "intermediate" saccules in the middle of the stacks, containing nicotinamide adenine dinucleotide phosphatase activity, c) one or two "last" saccules rich in thiamine pyrophosphatase activity on the trans or mature side, and d) continuing beyond the trans side, the GERL element displaying acid phosphatase activity. The latter is associated with profiles equally rich in acid phosphatase and tentatively considered to be prosecretory granules. Finally, the ectoplasm adjacent to Reichert's membrane displays large, acid phosphatase-containing structures tentatively considered to be secretory granules. Thus, the extensive rER network, the well-compartmentalized Golgi apparatus, and the presence of structures which may be prosecretory and secretory granules indicate that the endodermal cells are well-equipped for the secretion of the components of Reichert's membrane.

Laurie, G W; Leblond, C P; Martin, G R

doi: 10.1177/30.10.6752264pmid: 6752264

Antibodies to type IV collagen were linked with peroxidase and used for direct immunostaining of Reichert's membrane and the associated cells of the rat parietal yolk sac. Immunostaining was observed throughout the thickness of Reichert's membrane and within the endodermal cells arranged as a single layer on its inner side. The immunostaining of endodermal cells was mainly present in the cisternae of rough endoplasmic reticulum (rER) and in the Golgi apparatus, where it could occur in any saccule, but predominated in the GERL elements and associated prosecretory granule-like structures. Moreover, the secretory granule-like structures present in the ectoplasm next to Reichert's membrane were also immunostained. Finally, immunostaining was observed in multivesicular bodies and occasionally in secondary lysosomes. The antigenicity detected by immunostaining in Reichert's membrane is attributed to type IV collagen itself, whereas the antigenicity of endodermal cells is assigned to precursors of this collagen. It is proposed that initial precursors arise in rER cisternae, migrate to Golgi saccules, and pass to the GERL element, where they accumulate into prosecretory granules, which, perhaps by fusion with one another, become secretory granules. The secretory granules in turn migrate to the cell surface where they release their content, which becomes the type IV collagen of Reichert's membrane. Some diversion from this pathway may account for the immunostaining of multivesicular bodies and lysosomes.

Laurie, G W; Leblond, C P; Martin, G R; Silver, M H

doi: 10.1177/30.10.6752265pmid: 6752265

Reichert's membrane and the endodermal cells of the parietal yolk sac were examined for the presence of laminin antigenicity using anti-laminin antibodies and the peroxidase-antiperoxidase sequence. Immunostaining was observed through the full width of Reichert's membrane and within endodermal cells. In these cells immunostaining was observed in rough endoplasmic reticulum (rER) cisternae and Golgi apparatus. The Golgi staining could occur in any saccule, but predominated in components interpreted as the last saccule of the stack, the GERL element, and associated prosecretory granules. The secretory granules found in the ectoplasm were also immunostained. Finally, multivesicular bodies showed some staining. The immunostaining of Reichert's membrane indicates the presence of laminin itself, while that of rER cisternae and the Golgi apparatus is attributed to laminin precursors. Presumably the biosynthesis of laminin occurs along the usual protein pathway, that is, from rER through Golgi saccules and the GERL element to secretory granules, which release their content into Reichert's membrane. The laminin immunostaining of Reichert's membrane and endodermal cells is similar to that of type IV collagen. It is, therefore, likely that the two substances are processed and secreted simultaneously.

Lu, C L; Cantin, M; Seidah, N G; Chrétien, M

doi: 10.1177/30.10.6752266pmid: 6752266

Sections of the hypothalami and pituitary glands of normal (Sprague-Dawley) and homozygous diabetes insipidus (Brattleboro) rats were stained with antiserum to a human pituitary glycopeptide (HPGP) by using the immunohistochemical peroxidase-antiperoxidase method at the light microscopic level. Our results show in normal rats that immunoreactive HPGP was localized in the perikarya of the magnocellular neurons of the hypothalamus, in the posterior pituitary, and in the nerve fibers distributed in the median eminence (ME) and in the areas between the supraoptic nuclei (SON), paraventricular nuclei (PVN), and median eminence and also in the suprachiasmatic nuclei (SCN), a part of the parvocellular system. In the Brattleboro rats, however, no staining was found either in the hypothalami or pituitary glands. The present data strongly support our previous hypothesis that HPGP, a 39 residue glycopeptide isolated from human neurohypophysis, may be part of the precursor of arginine-vasopressin and its neurophysin II (Pro-NP-AVP).

Pignal, F; Maurice, M; Feldmann, G

doi: 10.1177/30.10.6752262pmid: 6752262

Immunoperoxidase localization of albumin and fibrinogen in rat liver was tested with perfusion or immersion fixation and saponin as a membrane permeabilizing agent. The distribution of albumin- or fibrinogen-containing hepatocytes was examined by light microscopy. Labeled antibody penetration was assessed by electron microscopy on transversely cut cryostat sections. Paraformaldehyde liver fixation by perfusion, followed by incubation of the sections with labeled antibodies together with saponin, demonstrated that albumin and fibrinogen were present in all hepatocytes; mainly in the Golgi apparatus and rarely in the endoplasmic reticulum, the ultrathin sections being labeled throughout their entire thickness. A constant labeling of the endoplasmic reticulum was obtained when saponin was added from the beginning of fixation. In the absence of saponin, albumin was seen in most of the hepatocytes but only at the periphery of the transverse sections, in a few Golgi apparatus, and in some parts of the endoplasmic reticulum; under this condition, fibrinogen was not visualized in the hepatocytes. Paraformaldehyde liver fixation by immersion showed the presence of albumin or fibrinogen in a few hepatocytes only, with irregular labeled antibody penetration. The use of saponin did not improve albumin and fibrinogen localization, except when the liver was poorly fixed. These results show that liver fixation by perfusion gives a homogeneous labeling of all the hepatocytes, whereas fixation by immersion leads to a heterogeneous labeling. Satisfactory results are obtained with saponin, which must be used to improve the penetration of labeled antibodies when the liver is fixed by perfusion. Saponin does not work when immersion is employed, at least under the conditions tested.

Wood, G W; Travers, H

doi: 10.1177/30.10.6813369pmid: 6813369

A high proportion of non-Hodgkin's lymphomas are neoplastic proliferations of B lymphocytes, and, as such, express integral membrane and/or cytoplasmic immunoglobulin (Ig). Because these cellular proliferations are monoclonal, the Ig of all neoplastic lymphocytes will have identical light chain type and idiotype. These tumors sometimes contain significant amounts of polyclonal Ig. In this study we demonstrate that the polyclonal non-B-lymphocyte-associated Ig may be removed by washing tissue at low pH to reveal either neoplastic B lymphocytes or neoplastic "null" lymphocytes. These observations should facilitate the application of immunohistology to the routine diagnosis of lymphoma.

Nemoto, N; Kawaoi, A; Shikata, T

doi: 10.1177/30.10.6182184pmid: 6182184

The occurrence of alpha-fetoprotein (AFP)-containing hepatocytes in 12 human embryos and fetuses ranging from 30-32 days of estimated ovulation age (Streeter's horizon XIV) to 16-17 weeks of estimated menstrual age was investigated using the direct (horseradish peroxidase (HRP)-labeled anti-human AFP horse IgG(Fab')2) and/or indirect immunoperoxidase methods. Under light microscopy, the AFP-containing hepatocytes were successfully demonstrated in paraffin-embedded liver tissues fixed with 4% paraformaldehyde(PFA)-picric acid solution containing 0.5% glutaraldehyde (GA). As a result of the studies of the human fetal livers at different developmental stages, only a small number of AFP-containing hepatocytes were initially identified immunohistochemically at the stage of Streeter's horizon XIX. Ultrastructural immunohistochemistry (the block-staining method using anti-human AFP horse IgG(Fab')2) revealed that the immunoreactive products against AFP were demonstrated on the ribosomal granules of the rough endoplasmic reticulum (rER), outer nuclear envelopes, free ribosomes, and Golgi's apparatus, and also in the cisternal lumina of the ER. No reactive products were noted in the nuclei or mitochondria. Our observations confirmed the presence of AFP-producing ability of the hepatocytes and the ultrastructural localization of the sites of protein synthesis in the early stage of development of human livers. Furthermore, we describe the extreme usefulness of the block-staining method for demonstrating the subcellular localization of AFP using HRP-labeled reduced anti-AFP antisera on liver tissues fixed with 4% PFA-picric acid solution containing 0.5% GA.

Danielson, K G; Ohi, S; Huang, P C

doi: 10.1177/30.10.6752263pmid: 6752263

An indirect immunoperoxidase staining technique was employed to localize the heavy metal binding protein, metallothionein, in rat liver and kidney. Immunostaining for metallothionein was observed in all hepatocytes and most renal tubular cells. This protein was not detectable, however, in cell types such as endothelial or connective tissue cells, indicating that metallothionein synthesis or accumulation is tissue- and cell-type specific. Hepatocytes from cadmium-exposed animals showed increased staining for metallothionein. The presence of metallothionein was also seen extracellularly within the liver sinusoids and renal tubules in both normal and cadmium-exposed animals, suggesting transport and excretion, respectively, of this protein.

Goldstein, D J

doi: 10.1177/30.10.7130668pmid: 7130668

Diffraction effects may have to be taken into account in microdensitometry when dealing with relatively dark specimens even an order of magnitude larger than the wavelength of light, and become progressively more important with smaller objects. According to geometrical optical theory, when scanning across the straight edge of a uniformly absorbing, semi-infinite object the distribution error per scan line is directly proportional to the diameter of the measuring-spot. Diffraction theory predicts similar results for measuring-spots larger than about 3 times the wavelength of light, but a significant error per scan line with very small or even infinitesimal measuring-spots. Diffraction theory further indicates that point absorbance measurements can be 95 + % accurate in the centers of 6.25, 2.5, and 2.0 microns diameter disks with absorbances respectively up to about 1.0, 0.39, and 0.25, but that this accuracy is unattainable with any object less than 1.25 microns in diameter. Scanning, integrating absorbance measurements are of somewhat lower accuracy than central point measurements with relatively large objects, e.g., they are only about 89% accurate with a 6 micron diameter object of absorbance 1.0. With very small objects, diffraction theory shows distribution error to be almost independent of the size of the scanning spot, and with an object of less than about 0.125 micron diameter the apparent integrated absorbance predicted by diffraction theory is effectively identical with that predicted by geometrical theory for an infinitesimal object scanned with a finite measuring-spot, i.e., it is the product of the object area and 0.4343 (1-It), where It is the true transmission. Scanning microdensitometry of objects of very low true absorbance is effectively free from distribution error. In practice, distribution error can be reduced by using an off-peak wavelength, by reducing the area illuminated, and by routine measurement and offsetting of apparent glare (some of which is actually due to diffraction). Little or nothing is to be gained by using a measuring-spot smaller than about one-quarter the wavelength of light.

Nieder, G L; Corder, C N

doi: 10.1177/30.10.6890080pmid: 6890080

Pyruvate and lactate are important energy sources for preimplantation embryos cultured in vitro. The purpose of this study was to determine in vivo levels of these substances in mouse oviductal tissues throughout the estrous cycle. Quantitative histochemical assays were developed to analyze these metabolites in submicrogram samples of freeze-dried ampullar and isthmic oviduct. The potential for other alpha-keto acids to interfere with the pyruvate assay was assessed and found to be minimal with this procedure. The importance of the collection method in maintaining in vivo metabolite levels was demonstrated by the marked changes observed with extended anoxia or pentobarbital anesthesia. Pyruvate levels at 3 hr postovulation (2.6 mmol X kg-1 dry weight) were higher than 12 hr before ovulation or 12 to 72 hr after ovulation (1.6 to 2.2 mol X kg-1) in both ampulla and isthmus. Lactate levels were significantly increased at 12 to 24 hr in the isthmus (28 mmol X kg-1) compared to other times during the cycle (9-19 mmol X kg-1). The observed levels of these metabolites may reflect changes in oviductal metabolism, induced by the hormone pattern of the estrous cycle, that promote the availability of needed energy substrates for the early embryo.