Journal of Histochemistry & Cytochemistry

Fahimi, H. Dariush; Baumgart, Eveline

doi: 10.1177/002215549904701001pmid: 10490450

The past decade has witnessed unprecedented progress in elucidation of the complex problems of the biogenesis of peroxisomes and related human disorders, with further deepening of our understanding of the metabolic role of this ubiquitous cell organelle. There have been many recent reviews on biochemical and molecular biological aspects of peroxisomes, with the morphology and cytochemistry receiving little attention. This review focuses on the state-of-the-art cytochemical techniques available for investigation of peroxisomes. After a brief introduction into the use of the 3,3′-diaminobenzidine method for localization of catalase, which is still most commonly used for identification of peroxisomes, the cerium technique for detection of peroxisomal oxidases is discussed. The influence of the buffer used in the incubation medium on the ultrastructural pattern obtained in rat liver peroxisomes in conjunction with the localization of urate oxidase in their crystalline cores is discussed, particularly since Tris-maleate buffer inhibits the enzyme activity. In immunocytochemistry, quantitation of immunogold labeling by automatic image analysis enables quantitative assessment of alterations of proteins in the matrix of peroxisomes. This provides a highly sensitive approach for analysis of peroxisomal responses to metabolic alterations or to xenobiotics. The recent evidence suggesting the involvement of ER in the biogenesis of “preperoxisomes” is mentioned and the potential role of preembedding immunocytochemistry for identification of ER-derived early peroxisomes is emphasized. The use of GFP expressed with a peroxisomal targeting signal for the investigation of peroxisomes in living cells is briefly discussed. Finally, the application of in situ hybridization for detection of peroxisomal mRNAs is reviewed, with emphasis on a recent protocol using perfusion-fixation, paraffin embedding, and digoxigenin-labeled cRNA probes, which provides a highly sensitive method for detection of both high- and low-abundance mRNAs encoding peroxisomal proteins.

Willingham, Mark C.

doi: 10.1177/002215549904701002pmid: 10490451

Josephsen, Kaj; Smith, Charles E.; Nanci, Antonio

doi: 10.1177/002215549904701003pmid: 10490452

Vimentin, an intermediate filament component, has been identified in many mesenchymal cells by a variety of LM and EM immunolabeling techniques. In our study, several tissue-processing conditions and monoclonal and polyclonal antibodies against vimentin were screened for immunostaining of rat incisor odontoblasts. Using postembedding colloidal gold immunocytochemistry, we were unable to detect any convincing vimentin antigenicity in these cells, but one of the monoclonal antibodies (V9-S) unexpectedly resulted in intense labeling over intra- and extracellular compartments that normally are strongly immunoreactive with anti-amelogenin antibodies. Blocking experiments showed that V9-S binding was competed by anti-amelogenin antibody. Immunoblots indicated that enamel proteins reacted with this anti-vimentin antibody after fixation with glutaraldehyde. These data suggest that the observed immunoreaction is directed against an epitope apparently created by crosslinking of enamel proteins during fixation. Although the labeling cannot be considered specific, it is nevertheless selective because it is very precisely localized over compartments containing enamel proteins and shows no binding to other calcified dental tissues, including dentin and bone. The V9-S antibody can therefore be used as a reliable probe to identify the presence and distribution of amelogenins in fixed tissues.

Skinner, Mhairi A.; MacLaren, Leslie A.; Wildeman, Alan G.

doi: 10.1177/002215549904701004pmid: 10490453

The 16-kD subunit of the vacuolar H+ -ATPase (V-ATPase), or ductin, is essential for the activity of this proton pump and has roles in intercellular communication and control of cell growth and differentiation. The V-ATPase is important for acidification-dependent degradation of tissue matrices through which some cell types move, and for pH regulation across some epithelial cell layers. Placentation involves intricate signaling, cell proliferation, and controlled invasion. We examined the distribution of three subunits of the V-ATPase in bovine trophoblast and endometrium at the time of implantation to determine the relationship of ductin expression to that of two other subunits, A (approximately 73 kD) and B (approximately 58 kD). Epithelial expression of all three subunits was observed, and in nonpregnant animals this expression was apical. As pregnancy proceeded, expression of all subunits became pericellular in luminal but not glandular epithelium, suggesting a redistribution of V-ATPase activity. The trophoblast expressed all three subunits during initial contact with the epithelium. In the stroma, ductin expression was reduced after implantation, and we discuss the possibility that ductin plays a role in the shifting communication between stromal and epithelial cells induced by embryo attachment.

Gao, Bo; Stieger, Bruno; Noé, Birgitta; Fritschy, Jean-Marc; Meier, Peter J.

doi: 10.1177/002215549904701005pmid: 10490454

In this study we investigated the distribution of a recently cloned polyspecific organic anion transporting polypeptide (Oatp2) in rat brain by nonradioactive in situ hybridization histochemistry and immunofluorescence microscopy. The results demonstrate that Oatp 2 is expressed in brain capillary and in plexus epithelial cells. At the blood-brain barrier (BBB), Oatp 2 expression could be co-localized with the endothelial marker vWF (von Willebrand factor) but not with the astrocyte marker GFAP (glial fibrillary acidic protein). In choroid plexus epithelial cells, Oatp 2 could be localized to the basolateral cell pole, whereas the first member of the Oatp gene family of membrane transporters to be cloned (Oatp 1) co-localized with the α1subunit of Na,K-ATPase at the apical plasma membrane domain. Because Oatp 1 and Oatp 2 have been previously shown to mediate transmembrane transport of a wide variety of amphipathic organic compounds, including many drugs and other xenobiotics, the histochemical localization of Oatp 2 at the BBB and of Oatp 1 and Oatp 2 in the choroid plexus imply a role for these transporters in the active exchange of amphipathic solutes between the blood, brain, and cerebrospinal fluid compartments.

Salisbury, Jeffrey L.; Lingle, Wilma L.; White, Richard A.; Cordes, Lynn E.M.; Barrett, Susan

doi: 10.1177/002215549904701006pmid: 10490455

We used a novel adaptation of methods for microtubule polymerization in vitro to assess the MTOC activity of centrosomes in frozen-sectioned tissues. Remarkably, centrosomes of tissue sections retain the ability to nucleate microtubules even after several years of storage as frozen tissue blocks. Adaptations of these methods allow accurate counts of microtubules from individual cells and the quantitative estimation the MTOC activity of the intact tissue. These methods can be utilized to characterize MTOC activity in normal and diseased tissues and in particular tissues at different stages of development.

Matsuzaki, Toshiyuki; Suzuki, Takeshi; Koyama, Haruko; Tanaka, Shigeyasu; Takata, Kuniaki

doi: 10.1177/002215549904701007pmid: 10490456

Aquaporins (AQPs) are membrane water channel proteins expressed in various tissues in the body. We surveyed the immunolocalization of AQP3, an isoform of the AQP family, in rat epithelial tissues. AQP3 was localized to many epithelial cells in the urinary, digestive, and respiratory tracts and in the skin. In the urinary tract, AQP3 was present at transitional epithelia. In the digestive tract, abundant AQP3 was found in the stratified epithelia in the upper part, from the oral cavity to the forestomach, and in the simple and stratified epithelia in the lower part, from the distal colon to the anal canal. In the respiratory tract, AQP3 was present in the pseudostratified ciliated epithelia from the nasal cavity to the intrapulmonary bronchi. In the skin, AQP3 was present in the epidermis. Interestingly, AQP3 was present at the basal aspects of the epithelia: in the basolateral membranes in the simple epithelia and in the multilayered epithelia at plasma membranes of the basal to intermediate cells. During development of the skin, AQP3 expression commenced late in fetal life. Because these AQP3-positive epithelia have a common feature, i.e., they are exposed to an environment of possible water loss, we propose that AQP3 could serve as a water channel to provide these epithelial cells with water from the subepithelial side to protect them against dehydration.

Douziech, Mélanie; Laberge, Gino; Grondin, Gilles; Daigle, Nathalie; Blouin, Richard

doi: 10.1177/002215549904701008pmid: 10490457

DLK/MUK/ZPK is a serine/threonine kinase that belongs to the mixed-lineage (MLK) subfamily of protein kinases. As is the case for most members of this family, relatively little is known about the physiological role of DLK/MUK/ZPK in mammalian cells. Because analysis of subcellular distribution may provide important clues concerning the potential in vivo function of a protein, an antiserum was generated against the amino terminal region of murine DLK/MUK/ZPK and used for localization studies in wild-type NIH 3T3 cells. Light microscopic immunocytochemistry experiments performed with the antiserum revealed that DLK/MUK/ZPK was specifically localized in a juxtanuclear structure characteristic of the Golgi complex. In support of this, treatment of cells with brefeldin A, a drug known to disintegrate the Golgi apparatus, caused disruption of DLK/MUK/ZPK peri-nuclear staining. Ultrastructural observation of NIH 3T3 cells also confirmed this localization, showing that most of the immunoreactivity was detected on membranes of the stacked Golgi cisternae. Consistent with localization studies, biochemical analyses revealed that DLK/MUK/ZPK was predominantly associated with Golgi membranes on fractionation of cellular extracts and was entirely partitioned into the aqueous phase when membranes were subjected to Triton X-114 extraction. On the basis of these findings, we suggest that DLK/MUK/ZPK is a peripheral membrane protein tightly associated with the cytoplasmic face of the Golgi apparatus.

Bergmann, Mathias; Grabs, Detlev; Rager, Günter

doi: 10.1177/002215549904701009pmid: 10490458

Dynamin I, a GTPase involved in the endocytic cycle of synaptic vesicle membranes, is believed to support axonal outgrowth and/or synaptogenesis. To explore the temporal and spatial patterns of dynamin I distribution in neuronal morphogenesis, we compared the developmental expression of dynamin with the expression of presynaptic membrane proteins such as SV2, synaptotagmin, and syntaxin in the chick primary visual pathway. Western blots of retina and tectum revealed a steady increase of synaptotagmin and syntaxin from embryonic Day 7 (E7) to E11, whereas for the same time frame no detectable increase of dynamin was found. Later stages showed increasing amounts of all tested proteins until the first postnatal week. Immunofluorescence revealed that SV2, synaptotagmin, and syntaxin are present in retinal ganglion cell axons from E4 on. In later stages, the staining pattern in the retina and along the visual pathway paralleled the formation and maturation of axons. In contrast, dynamin is not detectable by immunofluorescence in the developing retina and optic tectum before synapse formation. Our data indicate that, in contrast to the early expression of synaptotagmin, SV2, and syntaxin during axonal growth, dynamin is upregulated after synapse formation, suggesting its function predominantly during and after synaptogenesis but not in axonogenesis.

Ornberg, Richard L.; Woerner, B. Mark; Edwards, Dorothy A.

doi: 10.1177/002215549904701010pmid: 10490459

We describe a new light microscopic imaging system and method to perform high through put color image analysis on histological tissue sections. The system features a computer-controlled, random-access liquid crystal tunable filter and high-resolution digital camera on a conventional brightfield microscope. For any combination of stains, the method determines the spectral transmittance of each stain on the slide and selects two or more wavelengths at which the differential absorption between stain and counterstain is greatest and the exposure time is reasonably short. Flatfield corrected digital images at these wavelengths are acquired and divided to produce a gray scale ratio image. The ratio image is calculated such that the stained features of interest are highlighted above a uniform background and the counterstained features are highlighted below background. Image threshold procedures using either visual inspection or a threshold value determined by the image mean intensity and standard deviation are used to segment the stained features of interest for subsequent morphometry. Results are presented for peroxidase-AEC-labeled tumor tissue and trichrome-stained biomaterial implant tissues. In principle, the method should work for any combination of colored stains.