Journal of Histochemistry & Cytochemistry

Melvin, Neal R.; Sutherland, Robert J.

doi: 10.1369/jhc.2009.954164pmid: 19995945

Immunohistochemistry is a ubiquitous technique in histology. Often, the goal of such studies is the quantification of some parameter associated with a particular antigen. When used correctly, the optical disector offers a statistically relevant approach to achieve this goal without bias from cell size, shape, or orientation. This three-dimensional counting probe is virtually embedded within the depth of the tissue section, thus avoiding sampling near the cut surfaces of the section, where cells are often lost during the cutting and subsequent processing steps. It follows that the probability that a cell could be immunolabeled should be equal throughout the section depth to correctly employ the optical disector. In this report, we demonstrate that parameters commonly used in immunohistochemistry often leave the middle of the section unlabeled. Furthermore, the degree of incomplete penetration varies among antibodies but can be overcome in some cases by extending the incubation time of the secondary antibody. The detection of this phenomenon in immunofluorescence preparations and the implications of these findings for quantitative stereology using the optical disector are discussed.

Harada, Keita; Matsuoka, Hidetada; Fujimoto, Naohiro; Endo, Yutaka; Hasegawa, Yoshitaka; Matsuo, Akira; Kikuchi, Yuta; Matsumoto, Tetsuro; Inoue, Masumi

doi: 10.1369/jhc.2010.955575pmid: 20231739

The localization of the type-2 angiotensin II receptor (AT2) in the adrenal glands of rats, guinea pigs, bovines, and humans was examined at the mRNA and protein levels. PCR products for AT2 were detected in the adrenal cortices and adrenal medullae of all the mammals examined with an RT-PCR technique. Three different anti-AT2 antibodies (Abs), whose specificity was confirmed in our hands, recognized a 50-kDa protein in the adrenal glands of the four mammals, and this recognition was abolished by the preabsorption of an Ab with an antigen. Immunoblotting and immunohistochemistry revealed that the 50-kDa protein was expressed consistently and variably in the adrenal cortices and medullae of various mammals, respectively. We conclude that the 50-kDa AT2 is consistently expressed in the adrenal cortex in a wide variety of mammals.

Kollara, Alexandra; Brown, Theodore J.

doi: 10.1369/jhc.2010.955294pmid: 20354146

Human nuclear receptor coactivator 4 (NcoA4) amplifies the activity of several ligand-activated nuclear transcription factors, including the aryl hydrocarbon receptor (AhR) and androgen receptor (AR). Because these receptors exert important regulatory effects during development, with AhR ubiquitously expressed after embryonic day 9.5 (E9.5) and AR expressed from E12 onward, we examined NcoA4 expression in mouse embryos from E9.5 to E17.5. Full-length NcoA4 transcript was detected by RT-PCR at all embryonic stages and in all adult mouse tissues examined, although a novel splice variant was also detected. Western blot analysis indicated the expression of full-length NcoA4 protein, which was more highly expressed at later (E15.5–E17.5) embryonic stages. NcoA4 protein was also present at varying levels in all adult mouse tissues examined. A dynamic expression profile for NcoA4 during early development was indicated by immunohistochemistry in cardiac, hepatic, and lung tissue. Unlike human NcoA4, murine NcoA4 lacks an LXXLL motif, which has been implicated in the interaction with AR. Overexpression of murine NcoA4 augmented the transcriptional activity of AhR by 5-fold and AR by only 1.5-fold in COS cells. These studies demonstrate ubiquitous NcoA4 expression throughout development and suggest that this coactivator may play a role in modulating nuclear receptor activity, particularly that of the AhR, during development.

Wang, Changning; Popescu, Daniela C.; Wu, Chunying; Zhu, Junqing; Macklin, Wendy; Wang, Yanming

doi: 10.1369/jhc.2010.954842pmid: 20354147

We describe a novel fluorescent dye, 3-(4-aminophenyl)-2H-chromen-2-one (termed case myelin compound or CMC), that can be used for in situ fluorescent imaging of myelin in the vertebrate nervous system. When administered via intravenous injection into the tail vein, CMC selectively stained large bundles of myelinated fibers in both the central nervous system (CNS) and the peripheral nervous system (PNS). In the CNS, CMC readily entered the brain and selectively localized in myelinated regions such as the corpus callosum and cerebellum. CMC also selectively stained myelinated nerves in the PNS. The staining patterns of CMC in a hypermyelinated mouse model were consistent with immunohistochemical staining. Similar to immunohistochemical staining, CMC selectively bound to myelin sheaths present in the white matter tracts. Unlike CMC, conventional antibody staining for myelin basic protein also stained oligodendrocyte cytoplasm in the striatum as well as granule layers in the cerebellum. In vivo application of CMC was also demonstrated by fluorescence imaging of myelinated nerves in the PNS.

Kang, Lucia H.D.; Rughani, Agita; Walker, Matthew L.; Bestak, Rosa; Hoh, Joseph F.Y.

doi: 10.1369/jhc.2010.955419pmid: 20354144

We test the hypothesis that cat jaw satellite cells belong to a distinct lineage preprogrammed to express masticatory-specific isoforms of myosin heavy-chain (m-MyHC), myosin-binding protein-C (m-MBP-C), and tropomyosin (m-Tm) during myogenesis in vitro. A monoclonal antibody (MAb) against m-MyHC and MAbs raised here against cat m-MBP-C and m-Tm were used to stain cryostat sections of cat masseter muscle and cultured myotubes derived from satellite cells of cat temporalis and limb muscles, using peroxidase immunohistochemistry. MAbs against m-MBP-C bound purified m-MBP-C in Western blots. MAbs against m-Tm failed to react with m-Tm in Western blots, but reacted with native m-Tm in gel electrophoresis–derived ELISA. In cat masseter sections, MAbs against m-MyHC, m-MBP-C, and m-Tm stained all masticatory fibers, but not the jaw-slow fibers. Cat jaw and limb muscle cultures mature significantly more slowly relative to rodent cultures. However, at 3 weeks, all three MAbs extensively stained temporalis myotubes, whereas they apparently stained isolated myotubes weakly in cat limb and rat jaw cultures. We conclude that satellite cells of masticatory fibers are preprogrammed to express these isoforms during myogenesis in vitro. These results consolidate the notion that masticatory and limb muscle allotypes are distinct.

Jamison, James M.; Gilloteaux, Jacques; Perlaky, Laszlo; Thiry, Marc; Smetana, Karel; Neal, Deborah; McGuire, Karen; Summers, Jack L.

doi: 10.1369/jhc.2010.956284pmid: 20385787

Ascorbate and menadione (Apatone) in a ratio of 100:1 kills tumor cells by autoschizis. In this study, vitamin-induced changes in nucleolar structure were evaluated as markers of autoschizis. Human bladder carcinoma (T24) cells were overlain with vitamins or with culture medium. Supernatants were removed at 1-hr intervals from 1 to 4 hr, and the cells were washed with PBS and prepared for assay. Apatone produced marked alterations in nucleolar structure including redistribution of nucleolar components, formation of ring-shaped nucleoli, condensation and increase of the proportion of perinucleolar chromatin, and the enlargement of nucleolar fibrillar centers. Immunogold labeling of the nucleolar rRNA revealed a granular localization in treated and sham-treated cells, and immunogold labeling of the rDNA revealed a shift from the fibrillar centers to the condensed perinucleolar chromatin. Fibrillarin staining shifted from the fibrillar centers and adjacent regions to a more homogeneous staining of the entire nucleolus and was consistent with the percentage of autoschizic cells detected by flow cytometry. Because autoschizis entails sequential reactivation of DNase I and DNase II, and because the fibrillarin redistribution following DNase I and Apatone treatment is identical, it appears that the nucleolar and fibrillarin changes are markers of autoschizis.

Roussa, Eleni; Wittschen, Petra; Wolff, Natascha A.; Torchalski, Blazej; Gruber, Achim D.; Thévenod, Frank

doi: 10.1369/jhc.2010.955211pmid: 20385786

mCLCA1/2 are members of the CLCA protein family that are widely expressed in secretory epithelia, but their putative physiological role still awaits elucidation. mCLCA1/2 have 95% amino acid identity, but currently no specific antibody is available. We have generated a rabbit polyclonal antibody (pAb849) against aa 424–443 of mCLCA1/2. In HEK293 cells transfected with mCLCA1; pAb849 detected two specific protein bands at ×125 kDa and 90 kDa, representing full-length precursor and N-terminal cleavage product, respectively. pAb849 also immunoprecipitated mCLCA1 and labeled the protein by immunostaining. But pAb849 crossreacted with mCLCA3/4/6 despite ≤80% amino acid identity of the antigenic epitope. We therefore investigated the cellular localization of mCLCA1/2 in epithelial tissues, which do not express mCLCA3/4/6 (salivary glands, pancreas, kidney) or express mCLCA3/6 with known localization (mucus cells of stomach and small intestine; villi of small intestine). mCLCA1/2 mRNA and protein expression were found in both parotid and submandibular gland, and immunohistochemistry revealed labeling in parotid acinar cells, in the luminal membrane of parotid duct cells, and in the duct cells of submandibular gland. In exocrine pancreas, mCLCA1/2 expression was restricted to acinar zymogen granule membranes, as assessed by immunoblotting, immunohistochemistry, and preembedding immunoperoxidase and immunogold electron microscopy. Moreover, mCLCA1/2 immunolabeling was present in luminal membranes of gastric parietal cells and small intestinal crypt enterocytes, whereas in the kidney, mCLCA1/2 protein was localized to proximal and distal tubules. The apical membrane localization and overall distribution pattern of mCLCA1/2 favor a transmembrane protein implicated in transepithelial ion transport and protein secretion.

Larsen, Karen B.; Lutterodt, Melissa C.; Møllgård, Kjeld; Møller, Morten

doi: 10.1369/jhc.2010.955757pmid: 20354145

In rodents, the Otx2 gene is expressed in the diencephalon, mesencephalon, and cerebellum and is crucial for the development of these brain regions. Together with Otx1, Otx2 is known to cooperate with other genes to develop the caudal forebrain and, further, Otx1 is also involved in differentiation of young neurons of the deeper cortical layers. We have studied the spatial and temporal expression of the two homeobox genes OTX2 and OTX1 in human fetal brains from 7 to 14 weeks postconception by in situ hybridization and immunohistochemistry. OTX2 was expressed in the diencephalon, mesencephalon, and choroid plexus, with a minor expression in the basal telencephalon. The expression of OTX2 in the hippocampal anlage was strong, with no expression in the adjacent neocortex. Contrarily, the OTX1 expression was predominantly located in the proliferative zones of the neocortex. At later stages, the OTX2 protein was found in the subcommissural organ, pineal gland, and cerebellum. The early expression of OTX2 and OTX1 in proliferative cell layers of the human fetal brain supports the concept that these homeobox genes are important in neuronal cell development and differentiation: OTX1 primarily in the neocortex, and OTX2 in the archicortex, diencephalon, rostral brain stem, and cerebellum.