Journal of Histochemistry & Cytochemistry

Takizawa, Toshihiro; Suzuki, Kouki; Robinson, John M.

doi: 10.1177/002215549804601001pmid: 9742065

We demonstrate a fluorescent ultrasmall immunogold probe, FluoroNanogold (FNG), to be a versatile reporter system for immunocytochemical labeling of ultrathin cryosections. FNG-labeled molecules in the same ultrathin cryosections can be resolved by two imaging techniques (i.e., fluorescence and electron microscopy). Lactoferrin, a marker protein for the specific granules in human neutrophils, was employed as the target for FNG immunolabeling. The spatial resolution of the fluorescence signal from FNG-labeled specific granules was compatible with that of silver-enhanced gold signal from the same granules in electron microscopy. Our results confirm that FNG can be used as a probe for highresolution correlation between immunofluorescence and electron microscopy.

Gaub, Marie-Pierre; Lutz, Yves; Ghyselinck, Norbert B.; Scheuer, Isabelle; Pfister, Véronique; Chambon, Pierre; Rochette-Egly, Cécile

doi: 10.1177/002215549804601002pmid: 9742066

Apart from the retinoic acid nuclear receptor family, there are two low molecular weight (15 kD) cellular retinoic acid binding proteins, named CRABPI and II. Mouse monoclonal and rabbit polyclonal antibodies were raised against these proteins by using as antigens either synthetic peptides corresponding to amino acid sequences unique to CRABPI or CRABPII, or purified CRABP proteins expressed in E. coli. Antibodies specific for mouse and/or human CRABPI and CRABPII were obtained and characterized by immunocytochemistry and immunoblotting. They allowed the detection not only of CRABPI but also of CRABPII in both nuclear and cytosolic extracts from transfected COS-1 cells, mouse embryos, and various cell lines.

Malik, Zvi; Rothmann, Chana; Cycowitz, Tova; Cycowitz, Zwi J.; Cohen, Amos M.

doi: 10.1177/002215549804601003pmid: 9742067

Spectral morphometric characterization of typical chronic lymphocytic leukemia (B-CLL) cells vs normal small lymphocytes stained by May-Grunwald-Giemsa was carried out by multipixel spectral imaging. The light intensity (450–850 nm of 104 pixels) from nuclear domains of each stained cell was recorded and represented as light transmittance spectra and optical density. Transmitted light spectra of two nuclear domains were determined, one with low-intensity light transmittance (LIT) and the other with high-intensity light transmittance (HIT). A spectral library was constructed using the four transmitted light spectra representing the HIT and LIT domains of the normal human lymphocytes and the LIT and HIT domains of the CLL cells. The spectral library served to scan CLL lymphocytes from 10 cases of CLL and the lymphocytes of 10 healthy individuals. Each spectrally similar domain in the nuclei of the lymphocytes was assigned an arbitrary color. The morphometric analysis of the spectrally classified nuclei showed specific spectral patterns for B-CLL in 92% of the cells. The specific spectral characteristics of each of the two cell populations were also observed by their optical density light absorbance spectra. We propose that spectral morphometric analysis may serve as an additional diagnostic tool for detection of CLL lymphocytes in a hematological specimen.

Qu, Zhenhong; Kayton, Robert J.; Ahmadi, Proochista; Liebler, Janice M.; Powers, Michael R.; Planck, Stephen R.; Rosenbaum, James T.

doi: 10.1177/002215549804601004pmid: 9742068

We previously reported that mast cells (MCs) serve as a source of basic fibroblast growth factor (bFGF), a potent angiogenic and mitogenic polypeptide, suggesting that bFGF may mediate MC-related neovascularization and fibroproliferation. Unlike many other growth factors, bFGF lacks a classic peptide sequence for its secretion, and the mechanism(s) for its release remains controversial. Because MCs release a wide spectrum of bioactive products via degranulation, we hypothesized that MC degranulation may be a mechanism of bFGF release and used ultrastructural immunohistochemistry to test the hypothesis. We reasoned that if bFGF is released through degranulation, it should be localized to MC secretory granules. Human tissues with chronic inflammation and rat/mouse tissues with anaphylaxis were studied. In all tissue samples examined, positive staining (or immunogold particle localization) for bFGF in MCs was predominantly in the cytoplasmic granules. Moderate bFGF immunoreactivity was also found in the nucleus, whereas the cytosol and other subcellular organelles exhibited minimal immunogold particle localization. In contrast, no immunogold particle localization for bFGF was observed in lymphocytes or plasma cells. In rat/mouse lingual tissue undergoing anaphylaxis, immunogold particle localization for bFGF was found not only in swollen cytoplasmic granules but also in the extruded granules of MCs. Three different anti-bFGF antibodies gave similar immunogold particle localization patterns, whereas all controls were negative. These results provide morphological evidence suggesting that, despite the lack of a classic secretory peptide in its structure, bFGF is localized to the secretory granules in MCs and may be released through degranulation.

Loup, Fabienne; Weinmann, Oliver; Yonekawa, Yasuhiro; Aguzzi, Adriano; Wieser, Heinz-Gregor; Fritschy, Jean-Marc

doi: 10.1177/002215549804601005pmid: 9742069

We designed a protocol to improve the immunohistochemical analysis of human brain structures, which overcomes the limited detection sensitivity, high background, and intense autofluorescence commonly associated with human tissue. This procedure was evaluated by using antibodies against major GABAA receptor subunits (α1, α2, α3, γ2) in autopsy and surgical specimens. Tissue blocks were briefly fixed by immersion and pretreated with microwave irradiation in sodium citrate buffer. Immunoperoxidase staining revealed a marked enhancement of cell surface immunoreactivity and reduction of background in microwave- irradiated tissue, irrespective of its origin. For confocal laser scanning microscopy, immunofluorescence staining was optimized with the tyramide signal amplification (TSA) technique. This procedure not only dramatically increased the sensitivity for antigen detection but also totally suppressed autofluorescence, thus revealing the cellular and subcellular distribution of GABAA receptor subunits. A distinct neuron-specific expression pattern of the α-subunit variants was observed in cerebral cortex and hippocampal formation, along with widespread expression of the γ2-subunit. Of particular interest was the prominent α2- and α3-subunit staining on the initial axon segment of pyramidal neurons. This protocol represents a major improvement for high-resolution studies of human brain tissue aimed at investigating morphological alterations underlying neurological diseases.

Mino, Kazuto; Watanabe, Jun; Kanamura, Shinsuke

doi: 10.1177/002215549804601007pmid: 9742071

The effects of an inducer on expression of cytochrome P-450 (P-450) isoforms induced antecedently by another inducer are unknown. Thus, we examined the amount of phenobarbital (PB)-inducible P-450 isoforms (P-450 2B1/2B2) in hepatocytes from rats injected first with PB and then with 3-methylcholanthrene (MC) (PB+MC-treated animals) by quantitative immunohistochemistry. In addition, expression of P-450 2B2 mRNA was examined by in situ hybridization. In PB-treated animals, P-450 2B1/2B2 content increased in perivenular and midzonal hepatocytes. In PB+MC-treated animals, however, the PB-induced increase in 2B1/2B2 content was suppressed in perivenular hepatocytes but promoted in midzonal hepatocytes. The hybridization signal for P-450 2B2 mRNA appeared almost exclusively in perivenular hepatocytes after 24 hr of PB injection and disappeared after 48 hr of injection. In PB+MC-treated animals, however, strong hybridization signal was observed in midzonal and perivenular hepatocytes after 48 hr of PB injection. The promotion of the increase in P-450 2B1/2B2 content in midzonal hepatocytes in PB+MC-treated animals probably corresponds to the strong hybridization signal, whereas there appeared to be a divergence between the intensity of the signal and the content in perivenular hepatocytes. The results indicate that MC administration drastically influences the pattern of expression of P-450 isoforms induced by PB in perivenular and midzonal hepatocytes.

Johkura, Kohei; Usuda, Nobuteru; Liang, Yan; Nakazawa, Ayami

doi: 10.1177/002215549804601008pmid: 9742072

We studied the developmental changes in the localization of peroxisome-specific enzymes in rat kidney tissues from embryonic Day 16 to postnatal Week 10 by immunoblot analysis and immunohistochemistry, using antibodies for the peroxisomal enzymes catalase, d-amino acid oxidase, L-α-hydroxyacid oxidase (isozyme B), and enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase bifunctional protein. Peroxisomal enzymes were detected in the neonatal kidney by immunoblot analysis and their amount increased with kidney development. By light microscopic immunohistochemistry, they were first localized in a few proximal tubules in the juxtamedullary cortex of 18-day embryos. The distribution of proximal tubules positive for them expanded towards the superficial cortex with development. The full thickness of the cortex became positive for the staining by 14 days after birth. Peroxisomes could be detected by electron microscopy in structurally immature proximal tubules in 18-day embryos. Their size increased and the ultrastructure of subcompartments became clear with continuing development of proximal tubules. These results show that peroxisomal enzymes appear in the immature proximal tubules in the kidney of embryos and that the ultrastructure of the peroxisomes and localization of the peroxisomal enzymes develop along with the maturation of proximal tubules and kidney tissues.

Angermüller, Sabine; Künstle, Gerald; Tiegs, Gisa

doi: 10.1177/002215549804601009pmid: 9742073

Tumor necrosis factor (TNF) induces apoptotic death of hepatocytes in the galactosamine (GalN)-sensitized mouse liver after 5 hr. In our study, the most remarkable sign of the early stage of apoptosis was the focal rupture of the outer mitochondrial membrane. Parts of the inner membrane extended through the gap of the outer membrane, whereas the rest of the inner membrane still formed the cristae. This feature appeared in hepatocytes before chromatin condensation. With the diaminobenzidine technique for localization of cytochrome oxidase activity, the reaction product was detectable by light and electron microscopy. Ten percent of the hepatocytes were apoptotic, with condensed chromatin and high enzyme activity, 37% were pre-apoptotic, without chromatin condensation but high enzyme activity, and 53% had neither condensed chromatin nor a remarkable reaction product of cytochrome oxidase activity. Fas (APO-1, CD95) molecules on the plasma membrane of hepatocytes increased and were represented immunohistochemically in cells without chromatin condensation. DNA strand breaks were also detectable before chromatin aggregation. The results of this study indicate that mitochondria play a pivotal role in pre-apoptotic hepatocytes, together with an increase of the Fas molecule on the plasma membrane and with the occurrence of DNA strand breaks in the nucleus.

Arenas, María I.; Madrid, Juan F.; Bethencourt, Fermín R.; Fraile, Benito; Paniagua, Ricardo

doi: 10.1177/002215549804601010pmid: 9742074

The oligosaccharide sequences of glycoconjugates in the human normal epididymis and the nature of linkages were studied with lectin histochemistry. The usual terminal sequences of oligosaccharide side chains in epithelial cell secretions were Neu5Ac2,3Galβ 1,3GalNAc; SO4 Galβ1,3GalNAc; and Galβ1,4GlcNAc, and they were mainly found in O-linked glycoproteins. The lectin pattern of mitochondria-rich cells differed from that of principal cells.

Wu, Hong; Montone, Kathleen T.

doi: 10.1177/002215549804601011pmid: 9742075

Cortactin is a tyrosine kinase substrate that binds to filamentous actin. It represents a highly conserved family of perimembrane signaling proteins. The human homologue of cortactin is encoded by gene EMS1, which is amplified in some human breast, head, and neck carcinomas. This study shows that cortactin is preferentially localized to the apical surfaces of the polarized epithelium, specifically, to the terminal web of small bowel epithelium and to apical surfaces of the proximal renal tubules, thyroid follicles, and bronchiolar epithelium. Cortactin is also seen in cell and tissue types with actin-based contractile capacities, including smooth and striated muscle and myoepithelium.