Journal of Histochemistry & Cytochemistry

Hunyady, B; Krempels, K; Harta, G; Mezey, E

doi: 10.1177/44.12.8985127pmid: 8985127

The biotinyl-tyramide substrate of the horseradish peroxidase enzyme has been recently introduced to amplify immunohistochemical signals. We applied either fluorochromeor biotin-conjugated tyramine to improve the detection of different antigens in sections of rat stomach, pancreas, and hypothalamus. A ten- to 100-fold increase in staining efficiency was achieved, depending on the antibody, with either fluorescent or peroxidase detection systems. The amplification method was particularly useful for increasing a weak signal of conventional immunostaining caused by suboptimal tissue fixation. At a very low concentration of the primary antibody, the antigen can no longer be detected by a conventional fluorescent secondary antibody but is still detectable after amplification. When an antibody is used at this very low concentration and is detected by a fluorescent amplification method, another primary antibody, raised in the same host species, can be used and demonstrated with a different fluorochrome in subsequent conventional immunostaining of the same section. In this way it becomes possible to immunostain the same section with two different primary antibodies raised in the same host species. Samples for such double immunostaining are demonstrated here using pairs of monoclonal antibodies (to tyrosine hydroxylase and oxytocin) in the hypothalamus and polyclonal antibodies (to glucagon and neurofilament M) in sections of rat pancreas. Because in many cases the availability of antibodies is limited, the amplification method can be a quick and efficient tool for double immunostaining with antibodies from the same host species.

Poot, M; Zhang, Y Z; Krämer, J A; Wells, K S; Jones, L J; Hanzel, D K; Lugade, A G; Singer, V L; Haugland, R P

doi: 10.1177/44.12.8985128pmid: 8985128

Investigation of mitochondrial morphology and function has been hampered because photostable, mitochondrion-specific stains that are retained in fixed, permeabilized cells have not been available. We found that in live cell preparations, the CMXRos and H2-CMXRos dyes were more photostable than rhodamine 123. In addition, fluorescence and morphology of mitochondria stained with the CMXRos and CMXRos-H2 dyes were preserved even after formaldehyde fixation and acetone permeabilization. Using epifluorescence microscopy, we showed that CMXRos and H2-CMXRos dye fluorescence fully co-localized with antibodies to subunit I of cytochrome c oxidase, indicating that the dyes specifically stain mitochondria. Confocal microscopy of these mitochondria yielded colored banding patterns, suggesting that these dyes and the mitochondrial enzyme localize to different suborganellar regions. Therefore, these stains provide powerful tools for detailed analysis of mitochondrial fine structure. We also used poisons that decrease mitochondrial membrane potential and an inhibitor of respiration complex II to show by flow cytometry that the fluorescence intensity of CMXRos and H2-CMXRos dye staining responds to changes in mitochondrial membrane potential and function. Hence, CMXRos has the potential to monitor changes in mitochondrial function. In addition, CMXRos staining was used in conjunction with spectrally distinct fluorescent probes for the cell nucleus and the microtubule network to concomitantly evaluate multiple features of cell morphology.

Cook, T A; Mesa, K J; Gebelein, B A; Urrutia, R A

doi: 10.1177/44.12.8985129pmid: 8985129

Members of the dynamin superfamily are GTPases which have been shown to supportreceptor-mediated endocytosis in vivo and bind to growth factor receptor-associatedproteins in vitro. In acinar cells of the pancreas, receptor-mediated endocytosis isvery important for the recycling of membranes after secretory granule release.Therefore, characterization of the molecular machinery responsible for this processis critical for a better understanding of this phenomenon. In this study we sought todetermine the expression pattern of the endocytic GTPase dynamin II during pancreaticacinar cell differentiation in developing rat embryos and in dexamethasone-treatedAR42J cells using Western blot, Northern blot, and immunocytochemical analyses.During pancreatic development, dynamin immunoreactivity is almost undetectable untilday E17 but undergoes significant upregulation in acinar cells starting at E18. Inaddition, the levels of dynamin mRNA and protein in AR42J cells increaseapproximately threefold during dexamethasone-induced acinar differentiation. Theincrease in dynamin levels that occurs in both embryonic pancreatic cells anddexamethasone-treated AR42J cells correlates with the establishment of a moredifferentiated acinar phenotype. Therefore, these results suggest a potential rolefor dynamin in supporting receptor-mediated endocytosis in mature pancreatic acinarcells.

Hurskainen, T; Höyhtyä, M; Tuuttila, A; Oikarinen, A; Autio-Harmainen, H

doi: 10.1177/44.12.8985130pmid: 8985130

Cytotrophoblasts of early placenta invade the decidual membrane, gestationalendometrium, and spiral arteries during early pregnancy. Unlike tumor invasion, thisphysiological invasion is well controlled, although its molecular mechanisms arelargely unknown. We have previously shown that cytotrophoblasts synthesizesignificant mRNAs for 72-KD Type IV collagenase, laminin, and Type IV collagen,proteins implicated in extracellular matrix turnover and migration. In this study weused in situ hybridization and immunohistochemistry to investigate the mRNAexpression pattern of 92-KD Type IV collagenase and the matix metalloproteinaseinhibitors TIMP-1, TIMP-2, and TIMP-3 in early human placenta and decidual membrane.mRNAs for 92-KD Type IV collagenase, TIMP-1, TIMP-2, and TIMP-3 were found in thecells of cytotrophoblastic columns, the endothelial and fibroblastic stromal cells ofvilli, and the large decidualized cells of decidual membrane. TIMP-1 expression wasnotably accentuated in the fibroblasts of fibrotic villi. In the decidual membrane,the signals for 92-KD Type IV collagenase and TIMP-1 mRNA were particularly strongaround the glandular structures. The trophoblastic epithelium of villi and theepithelial cells of decidual glands showed a signal for 92-KD Type IV collagenase andTIMP-2, but not for TIMP-1 or TIMP-3. The coincidental expression of the proteolytic92-KD Type IV collagenase and inhibitors TIMP-1, TIMP-2, and TIMP-3 generally in thesame cells suggests that the activity of 92-KD Type IV collagenase, which isregulated by TIMPs, plays an important role in placental tissue organization and inthe invasion of trophoblastic cells into the uterine wall.

Henderson, M; Polewski, R; Fanning, J C; Gibson, M A

doi: 10.1177/44.12.8985131pmid: 8985131

This study used immunoelectron microscopic techniques to define the ultrastructurallocation of MAGP-1 on the fibrillin-containing microfibrils of the ocular zonule. Aspecific anti-MAGP-1 monoclonal antibody (MAb), 11B, was produced that did notcrossreact with fibrillin-1 or other microfibrillar proteins. MAb 11B was shown byimmunofluorescence to localize intensely to zonular tissue. Postembeddingimmunoelectron microscopy showed that MAGP-1 was associated with microfibrilsthroughout the zonule, with the exception of a narrow band of microfibrils at thejunction with the lens capsule. With preembedding labeling, the anti-MAGP-1 MAb wasfound to localize in a crossbanding pattern, at intervals of about 50 nm, tomicrofibrils throughout the zonule and along bundles of microfibrils in surroundingvitreous tissue. Rotary shadowing of isolated microfibrils showed a "beads on astring" morphology with a periodicity of about 50 nm. With immunogold labeling, theanti-MAGP-1 antibody specifically localized on the beads in a symmetrical manner.Occasionally two gold partides were attached to the same bead, suggesting thatmultiple MAGP-1 molecules were present in the structure. The results indicate thatMAGP-1 is intimately and regularly associated with the bead regions offibrillin-containing microfibrils. The findings are consistent with a majorstructural role for MAGP-1 in microfibril biology.

Gonzalez-Hernandez, T; Perez de la Cruz, M A; Mantolan-Sarmiento, B

doi: 10.1177/44.12.8985132pmid: 8985132

This study focused on two points concerning the histochemical and immunohistochemicaldetection of neurons that produce nitric oxide (NO): (a) the effect of fixation andother methodological parameters on the staining pattern of both NADPH-diaphorase(NADPH-d) histochemistry and nitric oxide synthase (NOS) immunohistochemistry, and(b) the possibility that neurons display immunoreactivity against NOS antiseraobtained from non-neuronal sources. Frontal sections of rat brains, fixed with 4%paraformaldehyde according to different protocols, were processed for single anddouble labeling using NADPH-d histochemistry and neuronal (nNOS), macrophagic(macNOS), and endothelial (eNOS) NOS immunohistochemistry. Our results show thatvariations in the fixative schedule, even within standard parameters, producequalitative and quantitative changes in NADPH-d labeling. The effect of fixative onweakly stained neurons is different from that on heavily stained neurons. In subfixedbrains, a large number of NOS-positive neurons lose their NADPH-d activity, whereasNOS immunolabeling remains unaltered. This finding may be particularly interesting inmorphological studies that compare NADPH-d activity under experimental conditionsthat can affect brain perfusion. On the other hand, many cortical and subcorticalneurons show macNOS immunoreactivity, most of it colocalized with nNOS.

Kamradt, J; Reichrath, J

doi: 10.1177/44.12.8985133pmid: 8985133

We analyzed immunohistochemically the expression of RAR proteins in basal cellcarcinomas (BCCs; n = 15) in situ. The labeling pattern for the different types ofRARs was compared with the staining pattern of the proliferation marker Ki-67 in thesame tumors. We found strong immunoreactivity for RAR-alpha and moderateimmunoreactivity for RAR-gamma in all BCCs analyzed, whereas no or very weak stainingfor RAR-beta protein was detected. In contrast to RAR-gamma, which revealed no oronly marginal differences in staining intensities, RAR-alpha immunoreactivity wasconsistently stronger in BCCs compared to adjacent unaffected epidermis. In general,labeling of BCCs for RAR-alpha and RAR-gamma was pronounced in cells of the palisadeand peripheral cells, whereas staining in the center of the tumors was heterogeneous.Eleven of the 15 BCCs analyzed revealed no visual correlation in comparing labelingpatterns for RAR-alpha and RAR-gamma with the labeling pattern for Ki-67. In fourspecimens, expression of RAR-alpha, RAR-gamma, and Ki-67 proteins was confined toperipheral tumor cells. Our findings indicate that (a) RAR-alpha and RAR-gammaproteins are, in contrast to RAR-beta, strongly expressed in BCCs; (b) expression ofRAR-alpha is upregulated in BCCs compared to keratinocytes of uninvolved epidermis;and (c) BCCs may be targets for potentially preventive or therapeutic treatment withRAR-alpha- or RAR-gamma-selective retinoic acid metabolites.

Appelt, D M; Kopen, G C; Boyne, L J; Balin, B J

doi: 10.1177/44.12.8985134pmid: 8985134

The purpose of this investigation was to identify and localize tissuetransglutaminase (TGase) within neurons from the hippocampi of normal agedindividuals and of those with confirmed Alzheimer's disease (AD). This enzyme may bea factor in the molecular mechanisms of neurodegeneration and formation of insolublemacromolecular complexes found in the neurons of normal aged and AD brain tissue. Anantibody made to the extracellular TGase, coagulation factor XIIIa, was found to bespecific for purified intracellular guinea pig liver tissue TGase. The specificityfor liver tissue TGase has enabled us to identify tissue TGase(s) within rathippocampal neurons and within neurons from normal aged and AD hippocampal tissues.Degenerating neurons from the AD hippocampus, compared to neurons from the normalaged hippocampus, exhibited increased immunoreactivity for TGase and demonstratedco-labeling for PHF1 and anti-TGase. Our results suggest that TGase may be associatedwith the neurofibrillary degeneration observed in AD, thereby implicating TGase as apotential factor in the pathogenesis of Alzheimer's disease.

Khoor, A; Gray, M E; Singh, G; Stahlman, M T

doi: 10.1177/44.12.8985135pmid: 8985135

Clara cell-specific 10-KD protein (CCSP) is an abundant product of nonciliatedbronchiolar epithelial (Clara) cells in the lung. We have determined thetemporal-spatial distribution of CCSP and its mRNA in developing human lung and inneonatal lung disease, using immunohistochemistry and in situ hybridization. CCSPimmunoreactivity was found in nonciliated bronchiolar epithelial cells from 12 weeksof gestation onward. Tracheal and bronchial epithelia showed positiveimmunoreactivity at each gestational week after 15 weeks and 14 weeks, respectively.CCSP mRNA was seen in the bronchial and bronchiolar epithelia from 16 weeks onwardand was detected in the trachea from 19 through 23 weeks of gestation. CCSPimmunoreactivity and mRNA were present in nonciliated single cells of bronchial andbronchiolar epithelia in fetuses and in infants with and without lung disease. CCSP-and CCSP mRNA-containing epithelial cells also formed dusters around neuroepithelialbodies (NEBs), especially at airway branch points, suggesting that NEBs and Claracells might interact during development and during pulmonary regeneration. Because ofevidence of overlapping of some but not all cells expressing CCSP, SP-A, and pro-SP-Bduring lung development, a common cell lineage is proposed, with subsequentdivergence of phenotypes.

Kameda, Y

doi: 10.1177/44.12.8985136pmid: 8985136

The sustentacular cells of the carotid body and the adrenal medulla of guinea pigs were studied by light and electron microscopic immunohistochemistry and compared with the Schwann or satellite cells of the peripheral nervous system. In the peripheral nervous system, neurons were immunoreactive for protein gene product (PGP) 9.5, whereas Schwann cells or satellite cells were immunoreactive for S-100 protein and vimentin. Vimentin immunoreactivity was detected on the intermediate filaments of the Schwann cells by post-embedding immunogold labeling. In the carotid body and the adrenal medulla, glomus cells or chromaffin cells were dosely enveloped by the sustentacular cells, which protruded long cytoplasmic processes and had some axons embedded in them, as in Schwann cells. The glomus cells or chromaffin cells expressed immunoreactivity for PGP 9.5, whereas the sustentacular cells expressed immunoreactivity for S-100 protein and vimentin. The sustentacular cells were characterized by the presence of abundant intermediate filaments on which immunogold particles for vimentin were densely located. From these results, it is concluded that the sustentacular cells closely resemble glial cells of the peripheral nervous system in immunohistochemical, ultrastructural, and also functional properties, and may belong to the glial lineage, originating from the neural crest.