Journal of Histochemistry & Cytochemistry

De Roe, C; Courtoy, P J; Baudhuin, P

doi: 10.1177/35.11.3655323pmid: 3655323

We prepared homogeneous populations of colloidal gold particles of various sizes. These were analyzed for size distribution and number of particles per unit volume. On exposure to increasing concentrations of insulin, myoglobin, protein A, peroxidase, serum albumin, galactosylated serum albumin, lactoferrin, transferrin, catalase, low-density lipoprotein, ferritin, and polymeric IgA, protein binding was a saturable process. Using serum albumin, we verified that a reversible equilibrium was reached within 15 minutes. Scatchard analysis of the interactions between all of these proteins and the gold particles resulted in a single component, linear relation. For a given particle size, the number of binding sites for various proteins was inversely proportional to their molecular weight. Conversely, when the size of particles was varied, the number of binding sites was directly proportional to the average area of each gold particle. All results are compatible with a monomolecular shell of protein surrounding the particle at saturation, the binding capacity being inversely proportional to the projection area of the protein. We present direct morphological evidence for this model. The affinity of the various proteins for the colloid also increased with molecular weight, and was not related to the protein isoelectric point. For globular proteins, the monomolecular shell model makes possible prediction of the number of molecules that will saturate a gold particle, if the average diameter of the gold particles and the molecular weight of the protein are known.

van der Loos, C M; Das, P K; Houthoff, H J

doi: 10.1177/35.11.2443555pmid: 2443555

For immunohistological analysis, simultaneous detection of multiple cellular epitopes, as compared to single staining of serial sections, is sometimes needed. Therefore, immunoenzyme triple-staining protocols were tested with polyclonal and monoclonal antibodies on tissue sections and cytospin preparations. Various immunoconjugates were used in different combinations of methods, of which not all proved to be suitable. Of the tested protocols, one yielded superior results for both monoclonal and polyclonal antibodies, with optimal preservation of their original avidity. The method consists of a combination of indirect, direct, and avidin-biotin complex technique. The three antigens can be distinguished clearly and selectively by the reaction products of the enzyme activities of beta-galactosidase (green), alkaline phosphatase (blue), and horseradish peroxidase (red).

Rosenquist, T H; McCoy, J R

doi: 10.1177/35.11.2443556pmid: 2443556

A direct Schiff reaction of elastic tissues has been known for many years, but the nature of the native aldehyde-rich components has not been clear. In this study, chicken, quail, and rat embryos and adult rat lung, aorta, and kidney were fixed in methacarn or in a formalin solution, embedded in paraffin, and sections of 8-10 micron obtained. Rehydrated sections were incubated for various periods in solutions of the enzymes chondroitinase ABC, clostripain, collagenase, elastase, heparatinase, hyaluronidase, subtilisin Carlsberg ("protease"), or trypsin, and in solutions of phosphomolybdic acid or sodium borohydride. After incubation, sections were placed, without prior oxidation, in Schiff's reagent, and were ultimately observed and photographed in transmitted light or with blue or green epifluorescence. A Schiff-positive substance was found, always and exclusively, in elastic tissues of the vasculature and lungs, which was hydrolyzed by the proteolytic enzymes to an extent that ranged from complete loss of Schiff reaction in minutes (trypsin) to no loss of Schiff reaction in 22 hr (clostripain). The Schiff-reactive protein preceded the time of appearance of elastin in the early embryos. We conclude that the aldehyde-rich protein responsible for this reaction is a harbinger of elastogenesis in vivo and speculate that it may represent the elastic microfibril or a component thereof.

Flavell, D J; Jones, D B; Wright, D H

doi: 10.1177/35.11.3309045pmid: 3309045

A mouse monoclonal antibody (MAC 387) with specificity for monocytes and tissue histiocytes was produced by immunization of a BALB/c mouse with peripheral blood monocyte components derived by affinity chromatography of detergent-solubilized monocyte material on Sepharose 4B coupled to rabbit anti-monocyte antibodies. MAC 387 strongly stained the cytoplasm of cells of the monocyte/macrophage series on paraffin sections after controlled trypsinization of sections. The antibody showed broad reactivity for a variety of tissue histiocytes, including infiltrating and reactive histiocytes, alveolar macrophages, Kupffer cells, follicle-center macrophages, splenic red pulp macrophages, tumor-infiltrating macrophages, sinus histiocytes, epithelioid giant cells (variably), and cases of histiocytosis X and dermatopathic lymphadenopathy. Molecular weight data obtained by Western blotting, immunoprecipitation, and immunoaffinity-purification revealed that the antigen was present in different forms in the monocyte and granulocyte. In the granulocyte, free alpha (Mr 12 KD) and beta (Mr 14 KD) chains expressing the MAC 387 epitope were found together with associations of one alpha and one beta chain linked by disulfide bonds to yield a heterodimer of Mr 26 KD. In the monocyte, free alpha and beta chains are not found, but instead the heterodimer and associations of two (Mr 56 KD) and four (Mr 112 KD) heterodimers are disulfide-linked together. This new monoclonal reagent should have particular value for identification of tissue histiocytes in routine paraffin sections and particularly for demonstration of histiocytes in malignant lymphomas.

Marks, S C; Grolman, M L

doi: 10.1177/35.11.3655324pmid: 3655324

Tartrate-resistant acid phosphatase (TRAP) has been used as a cytochemical marker for the cell mediators of bone resorption, osteoclasts and their mononuclear precursors. We have applied a cytochemical method for TRAP to study the dependence of the osteoclast-mediated bone resorption of tooth eruption on the dental follicle, a connective tissue investment of the developing tooth, by analyzing the TRAP activity of mononuclear cells in the dental follicle before and during pre-molar eruption in dogs. The percentage of TRAP-positive monocyte cells increases until mid-eruption, slightly preceding a previously demonstrated rise in numbers of osteoclasts on adjacent bone surfaces. These data suggest an ontogenetic relationship between follicular mononuclear cells and osteoclasts on adjacent alveolar bone surfaces during tooth eruption. However, because TRAP occurs in other tissues and is not an exclusive indicator of pre-osteoclasts, proof of their relationship will have to await application of more definitive techniques.

Spicer, S S; Erlandsen, S L; Wilson, A C; Hammer, M F; Hennigar, R A; Schulte, B A

doi: 10.1177/35.11.2443557pmid: 2443557

A wide range of tissues from three interfertile species of mice and an interspecific hybrid was examined with lectins conjugated to peroxidase to localize specifically glycoconjugates containing terminal alpha-N-acetylgalactosamine, alpha-galactose, and alpha-fucose, and the terminal disaccharide galactose-(beta 1----3)-N-acetylgalactosamine. This battery of lectins disclosed marked heterogeneity of glycoconjugates in different histological sites in a given animal and even between cells in a presumably homogeneous cell population within an organ. No variation with any lectin was observed between individuals of two closely related inbred strains of Mus domesticus at any specific histological or cytological site. In contrast, littermates of an outbred strain of Mus castaneus differed in binding of certain lectins at various sites, attesting to a genetic basis for individual variation. Hybrids between castaneus and domesticus mice also showed individual variation. Moreover, extensive differences between the mouse species were demonstrable with every lectin in glycoconjugates of stored secretions, Golgi cisternae, and apical or basolateral plasmalemma in many cell types. Totaling the differences in tabulated staining intensities for each possible species pair gave a measure of the overall extent of difference at 53 histological sites. According to this measure, the three species are about equally divergent from one another. Some differences between species appeared to depend on histological rather than histochemical variation, as, for example, a greater abundance of granular duct cells in the sublingual and submandibular glands in Mus hortulanus. Other differences were apparently derived from pathological change, as exemplified by casts and lymphoid infiltrates in kidney and structurally atypical submandibular gland lobules in Mus castaneus, and possibly by infiltrating cells in intestinal lamina propria and epithelium in Mus castaneus and hortulanus.

Decavel, C; Lescaudron, L; Mons, N; Calas, A

doi: 10.1177/35.11.3309046pmid: 3309046

A monoclonal antibody recently synthesized against dopamine (DA) was tested in rat and mouse brain sections after further treatment by PAP immunocytochemistry at the light and electron microscopic levels. Distribution of DA-immunoreactive cell bodies was examined in the substantia nigra (sn), the ventral tegmental area (vta), and the raphe nuclei. DA-immunoreactive fibers were investigated in two DA projection systems, the striatum and the septum. Many dopaminergic cell bodies were found in the sn and the vta. Some scattered DA neurons were encountered in the pars reticulata of the sn. The dorsal raphe and linearis raphe nuclei displayed sparse immunoreactive neurons and a dense plexus of DA fibers. Immunoreactive fibers were observed in the entire striatum, more dense in the ventral part. In the septum, immunonegative neurons were outlined by thin DA fibers in synaptic contact with their somata or dendrites. According to our observations, this DA monoclonal antibody seems to be a selective and sensitive tool for studying the dopaminergic neuronal circuitry at both histological and ultrastructural level.

Sklarew, R J; Pertschuk, L P

doi: 10.1177/35.11.2821106pmid: 2821106

A Quantimet 720D Image Analysis System has been programmed for light microscopic evaluation of the nuclear estrogen receptor distribution in frozen sections of human breast cancer stained by the peroxidase-antiperoxidase method using monoclonal antibodies to estrogen receptor protein (ER). This method provides precise criteria for distinguishing ER-positive and -negative cells and a sensitive and reproducible means for densitometric quantification of the staining patterns. Although imaging sequence and graphic analysis are automated by computer programs, light pen interaction provides supervision of feature selection. Imaging of the immunocytochemical assay (ER-ICA) in 50 patients revealed marked heterogeneity of nuclear estrogen receptor concentration varying over a nearly 100 fold concentration range. Various ER concentration patterns were evident: (I) distributions with a single peak (CV = 5%) present at various concentration levels; (II) bimodal distributions, revealing co-existent ER-positive and ER-negative subpopulations; (III) multimodal distributions with a number of resolvable concentration peaks; and (IV) highly skewed distributions with or without discernible peaks, frequently extending over the entire concentration range. Statistical methods of de-convolution were applied to determine the frequency and ER concentration characteristics of component subpopulations in the mosaic cases and for resolving the proportion of ER-positive and -negative cells. An approach for evaluating nuclear ER content in conjunction with ER concentration patterns in individual patients revealed whether spread in the ER concentration distribution resulted from differences in nuclear ER content or from variability in nuclear volume distribution.

Vorbrodt, A W

doi: 10.1177/35.11.3655325pmid: 3655325

An attempt was made to demonstrate the anionic sites on the endothelial cell (EC) surfaces of mouse brain micro-blood vessels (MBVs) after embedding of tissue samples in hydrophilic media: Lowicryl K4M, LR White, and Polyamph-10. As a cationic probe, poly-L-lysine-gold complex (PLG), prepared according to the procedure of Skutelsky and Roth (J Histochem Cytochem 34:693, 1986), was used. In ultra-thin sections of brain samples embedded in Lowicryl K4M and LR White, the anionic sites were demonstrated in the entire cross-section of the vessel wall. After embedding in Polyamph-10, however, the anionic sites could not be detected. Brain capillaries, representing blood-brain barrier type MBVs, showed polar distribution of anionic sites, evidenced by more intense labeling of luminal than of abluminal plasma membrane of the EC. Some differences in labeling of ECs and of basement membrane in arterioles and venules were also noted. The use of cationic gold and the ultra-thin sections of tissue samples embedded in hydrophilic media (Lowicryl K4M and LR White) seems to be a promising new method for detection of anionic constituents located on both luminal and abluminal surfaces of the EC, in the basement membrane, and in other components of the vessel wall.

Boffa, M C; Burke, B; Haudenschild, C C

doi: 10.1177/35.11.2821107pmid: 2821107

The protein C anticoagulant system is mediated by thrombin and is highly accelerated by thrombomodulin. We studied the distribution of thrombomodulin antigen (TM Ag) in the rabbit using an affinity-purified antibody raised in a goat against rabbit thrombomodulin. The preservation of TM Ag was highly dependent on immediate fixation of the surface on which it is located. TM Ag was found on the endothelium of the entire vasculature, whereas it was absent from all connective tissue, smooth and striated muscle, secretory epithelia, cartilage, bone, neural tissue, and all parenchyma examined. A new finding was the presence of TM Ag on nonvascular surfaces of body cavities (the mesothelia of pleura, pericardium, and peritoneum, the synovial membrane, and the arachnoid enveloping the central nervous system). By use of a functional assay, TM activity was recovered in buffered saline/detergent solution which was either injected into the intraperitoneal cavity of rabbits in vivo or incubated with the surface of the arachnoid in vitro. These findings extend the importance of anticoagulant mechanisms to the systems of slowly circulating fluids, in which they might be required for maintenance of the flow, and to mesothelial cavities, in which they could be necessary for preventing adherence between the surfaces, in conditions associated with pathological exudation.