Journal of Histochemistry & Cytochemistry

McMillan, P J; Leverenz, J B; Poorkaj, P; Schellenberg, G D; Dorsa, D M

doi: 10.1177/44.11.8918895pmid: 8918895

Mutations in the STM2 gene cause familial Alzheimer's disease (AD) in Volga Germans.To understand the function of this protein and how mutations lead to AD, it isimportant to determine which cell types in the brain express this gene. In situhybridization histochemistry indicates that STM2 expression in the human brain iswidespread and is primarily neuronal. In addition, STM2 mRNA is expressed in a cellline with neuronal origins. Quantification of the level of expression of the STM2message in the basal forebrain, frontal cortex, and hippocampus reveals a significantdecrease in AD-affected subjects compared to normal age-matched controls. These datasuggest that downregulation of neuronal STM2 gene expression may be involved in theprogression of AD.

Masunaga, T; Shimizu, H; Ishiko, A; Tomita, Y; Aberdam, D; Ortonne, J P; Nishikawa, T

doi: 10.1177/44.11.8918896pmid: 8918896

Laminin-5 (kalinin/nicein/BM600) is a component of the epidermal basement membranezone. Previous en bloc pre-embedding immunogold electron microscopy (EM) has shownthat the epitope of GB3, a monoclonal antibody against laminin-5, is present in thelamina lucida (LL). However, precise localization of the entire laminin-5 moleculewas unclear because of uneven and limited penetration of gold-labeled antibody inpre-embedding immunolabeling. In addition, the location of the GB3 epitope may notdirectly represent the location of the laminin-5 moelcule itself. To elucidate theprecise ultrastructural distribution of the entire laminin-5 molecule, we usedpolyclonal antibodies against different sites of laminin-5. Dual stainingimmunofluorescence with anti-laminin-5 and anti-melanocyte antibodies andimmunoperoxidase EM showed that laminin-5 was present only beneath keratinocytes andnot beneath melanocytes. Both cryoultramicrotomy and postembedding immunogold EMdemonstrated that laminin-5 was localized to the lamina densa (LD) and the lower LL,with major labeling beneath the hemidesmosome. Quantitative analysis showed that67-69% of gold particles were distributed to the LD and 88-90% were distributedbeneath the hemidesmosome. Our results indicate that laminin-5 is localized mainly tothe LD and partially to the lower LL, and is associated predominantly withhemidesmosomes.

Ogawa, A; Kurita, K; Ikezawa, Y; Igarashi, M; Kuzumaki, T; Daimon, M; Kato, T; Yamatani, K; Sasaki, H

doi: 10.1177/44.11.8918897pmid: 8918897

Heterogeneity of zonal hepatocytes is important to elicit specific liver function. Weinvestigated the distribution of glucose transporter 2 (GLUT-2) in normal rat liverby immunostaining and Northern blot analysis. GLUT-2 stained by immunohistochemistrywas distributed predominantly in the periportal hepatocytes and gradually thinnedtowards the perivenous zone. Ultrastructural immunostaining of GLUT-2 showed that itwas localized on microvilli of the sinusoidal plasma membrane of hepatocytes but noton the basolateral plasma membrane. Consistent with the distribution of GLUT-2protein, the level of GLUT-2 mRNA in periportal hepatocytes was 1.9-fold higher thanin perivenous hepatocytes selectively isolated by the differential isolationtechnique. In addition, the mRNA level of phosphoenolpyruvate carboxykinase, one ofthe key enzymes of gluconeogenesis, was also twofold higher in the periportalhepatocytes. These results suggest that GLUT-2 contributes to the functionaldifference between periportal and perivenous hepatocytes in glucose metabolism of theliver.

Sugiyama, T; Yamamoto-Hino, M; Wasano, K; Mikoshiba, K; Hasegawa, M

doi: 10.1177/44.11.8918898pmid: 8918898

We investigated the immunohistochemical localization of inositol 1,4,5-trisphosphatereceptor (IP3R) Types 1, 2, and 3 in rat airway epithelium using the monoclonalantibodies KM1112, KM1083, and KM1082 specific for each type of IP3R. The epitheliumfrom trachea to distal intrapulmonary airways (bronchioles) showed positiveimmunoreactivity for all types of IP3R. However, cell type as well as subcellularsite immunoreactivity for each type of IP3R varied. IP3R Type 1 was found only in theapical thin cytoplasmic area of ciliated cells throughout all airway levels. IP3RType 2 was exclusively localized to the entire cytoplasm of ciliated cells from thetrachea to bronchioles. IP3R Type 3 was expressed mainly in the supranuclearcytoplasm not only of ciliated cells at all airway levels but also in Clara cells ofthe bronchiolar epithelium. Double fluorescent staining using combinations of KM1083and Wisteria floribunda lectin or anti-rat 10-KD Clara cell-specific protein antibodyconfirmed that the IP3R Type 2-positive cells were neither seromucous cells nor Claracells. These results indicate that the expression of three types of IP3Rs indifferent cell types and subcellular sites may reflect diverse physiologicalfunctions of IP3Rs within airway epithelial cells. The double staining studiessuggested that the anti-IP3R Type 2 monoclonal antibody KM1083 would be a specificcell marker for ciliated cells of the airway epithelium.

Matovcik, L M; Maranto, A R; Soroka, C J; Gorelick, F S; Smith, J; Goldenring, J R

doi: 10.1177/44.11.8918899pmid: 8918899

The Type 3 inositol 1,4,5-trisphosphate (InsP3) receptor is expressed at high levelsin gastrointestinal tissues. This receptor has 16 potential phosphorylation sites forcalcium/calmodulin-dependent protein kinase II (CaM kinase II). To determine if theType 3 InsP3 receptor is likely to be a physiologic substrate for CaM kinase II,localizations of the Type 3 InsP3 receptor and CaM kinase II were compared in tissuesof the gastrointestinal tract. Cellular and subcellular localizations were determinedby immunofluorescence microscopy in rat intestine, pancreas, and stomach, and inisolated rabbit gastric glands. Both proteins were found in the apical region ofintestinal enterocytes, pancreatic acinar cells, and gastric parietal, chief, andsurface mucous cells. CaM kinase II was found throughout the entire intracellularcanalicular F-actin domain of parietal cells, whereas the type 3 InsP3 receptor wasrestricted to the neck region. Thus, in several gastrointestinal tissues the Type 3InsP3 receptor is specifically localized to a portion of the apical cytoskeletaldomain in which resides the calcium-responsive effector CaM kinase II.

Reeves, J R; Going, J J; Smith, G; Cooke, T G; Ozanne, B W; Stanton, P D

doi: 10.1177/44.11.8918900pmid: 8918900

The relationship between expression of the c-erbB-2 proto-oncogene and the biology ofbreast cancer has been investigated widely, most studies using immunohistochemistryin formalin-fixed, paraffin-embedded tissues. This technique is at bestsemiquantitative and there is a high degree of interstudy variability because of itssubjective nature and poor methodological standardization. The relationship betweenthe levels of expression and biology can be examined thoroughly only with anaccurately quantitative technique. We have developed a radioimmunohistochemical assayto measure p185(erbB-2) in tissue biopsy specimens. The method involves incubatingfrozen sections with 125I-labeled monoclonal antibody, microautoradiograpy, and graincounting with image analysis. Sections of cell pellets with known c-erbB-2 levels areprocessed with each batch of samples as internal calibration standards. We havequantified c-erbB-2 expression in 60 breast carcinomas and compared the results withconventional immunohistochemistry. Radioimmunohistochemistry measured receptor levelsthroughout the range of expression in breast carcinomas, whereas conventionalimmunohistochemistry detected the protein only in the highest expressing tumors. Thequantitative, objective data produced by radioimmunohistochemistry allow a morethorough evaluation of the relationship between c-erbB-2 expression and tumorbiology. This technique may have applications in other fields where quantitative datais required and relevant monoclonal antibodies are available.

Funato, H; Yoshimura, M; Ito, Y; Okeda, R; Ihara, Y

doi: 10.1177/44.11.8918901pmid: 8918901

Here we report on the presence of proliferating cell nuclear antigen (PCNA) in humanleptomeninges from 35 normal subjects with ages ranging from 57 to 94 years. Strongimmunoreactivity with PC10 (a monoclonal antibody to PCNA) was detected in the nucleiof meningothelial cells, smooth muscle cells of leptomeningeal vessels, and ependymalcells. An immunoblot of leptomeningeal homogenate with PC10 showed the presence of asingle band at 35 KD, the expected molecular mass of PCNA. Ki-67, another marker forcell proliferation, was undetectable in human leptomeninges. These observations pointto isolated PCNA expression in tissue in which cells are not activelyproliferating.

Antonio, C; González-García, J M; Page, J; Suja, J A; Stockert, J C; Rufas, J S

doi: 10.1177/44.11.8918903pmid: 8918903

We analyzed first-metaphase meiotic chromosomes of the grasshopper Chorthippusjucundus by two different methods, i.e., a silver impregnation technique and theosmium tetroxide-p-phenylenediamine (Os-PPD) procedure. The former was applied onsquashed testes previously fixed in ethanol-acetic acid, whereas for Os-PPD thematerial was not subjected to any previous extraction treatment but was fixed inOsO4, treated with PPD, and embedded in Epon 812. Both techniques revealed chromatidcores and kinetochores regardless of the processing of the material (squashed orsectioned). Unstained Os-PPD sections were analyzed by light microscopy andtransmission electron microscopy (TEM). The Os-PPD technique provided a high contrastof chromatid cores and kinetochores in relation to the chromatin, which revealed alow electron density. To determine the Os-PPD reaction mechanism, the PAS procedure,as well as scanning electron microscopy (SEM) backscattering and SEM X-raymicroanalysis, was performed on sections. By use of the Os-PPD-PAS procedure, glycolgroups formed by oxidation of osmium bound to aromatic substrates were detected inchromatid cores and kinetochores by brightfield and fluorescence microscopy. A high Zcontrast was detected in these structures by backscattered electron imaging. SEMX-ray microanalysis showed osmium and phosphorus to be the main elements present onthe chromatid cores. Taking into account the known reactivity of OsO4 and the presentresults, the possible participation of nucleic acids as well as proteins in theOs-PPD reaction mechanism and in the composition of chromatid cores and kinetochoresis discussed.

Katsahambas, S; Hearn, M T

doi: 10.1177/44.11.8918904pmid: 8918904

In mated sows, the level of placental vascularization has a direct effect on fetalgrowth and litter birth weight. Vascularization of the endometrium and uterus underthe control of various polypeptide growth factors is an important early stage in thisprocess. Basic fibroblast growth factor (FGF-2), a polypeptide distributed throughoutthe mesodermal and neuroectodermal tissues of many species, is a vascular endothelialcell mitogen in vitro and has been implicated in neovascularization and wound healingin vivo. As part of our studies of the distribution of FGF-2 in uterine tissue andits role in placental development and embryo implantation, the localization andchanges in the abundance of porcine FGF-2 mRNA in the uterus of mated and unmatedgilts were investigated by in situ hybridization procedures. These procedures werebased on the use of [alpha35S]-dATP-labeled oligonucleotide probes and a novel set ofdigoxigenin-labeled oligonucleotide probes generated by reversetranscriptase-polymerase chain reaction (RT-PCR) methods and anti-sense labelingstrategies from the corresponding mRNA templates. With these in situ hybridizationprocedures, porcine FGF-2 mRNA was localized during the first 30 days of pregnancy tospecific tissue areas in the porcine uterus comprising glandular and luminalepithelial cells and stromal cells of both the stratum functionalis and stratumbasalis regions of the endometrium, and within the smooth muscle of myometrium andthe associated blood vessels. However, no significant increase in the level of FGF-2mRNA within these tissues was detected during these early stages of pregnancy orduring the estrous cycle of unmated gilts. These distribution and abundance patternsare only partially compatible with other recent observations suggesting a possiblerole for changing levels of the mature polypeptide form of FGF-2 in the reproductivetract of sows during the early stages of pregnancy.

Jacobsson, B; Bernell, P; Arvidsson, I; Hast, R

doi: 10.1177/44.11.8918905pmid: 8918905

We used peripheral blood (PB) and bone marrow (BM) smears in the development of twomethods based on cytomorphology and esterase cytochemistry in combination withfluorescence in situ hybridization (FISH). The first method involvesphotodocumentation of May-Grünewald-Giemsa (MGG)-stained cells, followed bydestaining in methanol-acetic acid, fixation in paraformaldehyde, and digestion withprotease and RNAse before FISH using alpha-satellite probes that specify chromosomesX, 7, 8, and 17. On average, two hybridization signals were seen in 94.5% of disomicBM cells. The hybridization sensitivity was found to vary, however, both amongmorphologically defined hematopoietic cell lineages and among differentation levelswithin a lineage. In the second method, an esterase staining technique was followedby the same treatment as for MGG-stained cells. The esterases and FISH signals couldbe simultaneously visualized and the method was found suitable for rapid screening ofin situ signals in cytochemically defined granulocytes and lymphocytes but not inmonocytes. The combined methods proved very useful in elucidating the clinicalsignificance of chromosomal abnormalities seen in two cases of leukemia.