bluuebunny/crossref_metadata_2025 huggingface dataset:
Distribution and lateral mobility of the urokinase-receptor complex at the cell surface.
Authors: Myöhänen H T , Stephens R W , Hedman K , Tapiovaara H , Rønne E , Høyer-Hansen G , Danø K , Vaheri A
Published: April 2011

Pro-urokinase (pro-uPA) and activated uPA are confined to focal adhesions and cell-cell contacts. We studied the distribution of the uPA receptor (uPAR) on human fibroblasts (HES) and rhabdomyosarcoma (RD) cells by immunofluorescence and immunoelectron microscopy. Two monoclonal antibodies (MAb) utilized were against uPAR: MAb R4, which reacts with occupied and unoccupied uPAR, was concentrated at focal adhesions; MAb R3 reacting with unoccupied receptor stained cell surfaces diffusely. MAb R4 stained cell-cell contacts, tips of microspikes, and co-localized with vinculin. Of the matrix and integrin components tested, alpha v beta 3 integrin was found at focal adhesions but more centrally than uPAR. Since uPAR is anchored to the plasma membrane through a GPI lipid, we studied its mobility by antibody-induced clustering. This revealed that unoccupied uPAR was relatively mobile; MAb R3 redistributed it to clusters. In contrast, uPAR R4 and uPA antibodies at the focal contact sites remained mostly within focal contacts. Addition of exogenous uPA resulted in loss of R3 staining and increase of uPA in focal adhesions. These results suggest that occupancy of the receptor with uPA is associated with localization to cell contact sites and restricted lateral mobility.
Localization of MOC-1 cell surface antigen in small-cell lung carcinoma cell lines: an immunohistochemical and immunoelectron microscopic study.
Authors: Speirs V , Eich-Bender S , Youngson C R , Cutz E
Published: April 2011

Expression of cell surface antigens of the neural cell adhesion molecule (N-CAM) class was recently shown to be shared by both fetal and neoplastic neuroendocrine cells, including those of the lung. We investigated the expression and localization of MOC-1 antigen on small-cell (neuroendocrine) lung carcinoma cell lines with immunohistochemical methods at the light (LM) and electron microscopy (EM) level and by Western blot. At LM level, using monoclonal antibody (MAb) MOC-1 with the ABC method and immunofluorescence, positive staining was observed on surfaces of cells from all tumor lines examined. Strongest immunostaining was found on cell surfaces of pulmonary small-cell carcinoma-derived cell line NCI-H69 with the majority of cells showing positive staining. An adherent variant of NCI-H69 cell line, H69V, exhibited positive staining in about 60% of cells, whereas only occasional cells of NCI-H727 cell line derived from pulmonary carcinoid tumor were positive for MOC-1 antigen. Western blot analysis confirmed these findings, showing a strong MOC-1-specific band in cell extracts of NCI-H69, with weaker band densities for H69V and NCI-H727. Immunoelectron microscopy (IEM) revealed that MOC-1 was not uniformly distributed on the outer surface of plasma membrane; immunogold particles appeared concentrated in areas of thick cell surface "fuzz" coating, surface microvilli, and in areas of cell-cell contact. In some cells, areas of plasma membrane invaginations and a few intracytoplasmic vesicles were also labeled, suggesting endocytosis. Surface labeling for SEM confirmed the finding of more dense labeling over the microvilli, cell membrane folds, and in areas of cell-cell contact. The cell lines derived from pulmonary neuroendocrine cell tumors can provide a useful model to study the role and function of neural adhesion molecules in pulmonary neoplasia and during lung development.
Expression of myosin heavy chain isoforms in developing human muscle spindles.
Authors: Pedrosa-Domellöf F , Thornell L E
Published: April 2011

We studied serial sections of human fetal limb muscles (10-25 weeks of gestation) by light microscopic (LM) immunocytochemistry, using specific antibodies against slow-tonic, slow-twitch, fetal, embryonic, and alpha-cardiac myosin heavy chain (MHC) isoforms, neurofilament protein, laminin, and myomesin. One set of the first-generation myotubes expressed slow-tonic MHCs, and slow-twitch, fetal, and embryonic MHCs from the tenth week of gestation. These primary myotubes were identified as developing nuclear bag fibers. Second-generation myotubes in close apposition to the primary nuclear bag myotubes initially expressed only fetal and embryonic MHCs. One or more of these secondary myotubes acquired expression of slow-tonic and slow-twitch MHCs and gave rise to nuclear bag fibers. Most of the nuclear bag precursors expressed alpha-cardiac MHC. The secondary myotubes that expressed fetal and embryonic MHC but not slow-tonic, slow-twitch, or alpha-cardiac MHCs gave rise to the nuclear chain fibers. This study shows that different populations of fiber precursors, each with a unique sequence of MHC expression, gave rise to the nuclear bag and chain fibers, despite the presence of a common afferent nerve, from the early stages.
In situ hybridization and cytochemistry: localization of mRNA at stained neuromuscular junctions with 33P-labeled probes.
Authors: Liu E , Salpeter M M
Published: April 2011

We modified the Karnovsky and Roots method of staining sites of acetylcholinesterase (AChE) activity at neuromuscular junctions (NMJs) to survive the lengthy, multiple steps of in situ hybridization and autoradiography. When the original method of Karnovsky and Roots is used to identify the muscle endplates, the stain does not survive the in situ hybridization procedures and association of mRNA to specific endplates can be inferred only indirectly. The successful modification involves secondary staining with diaminobenzidine (DAB) and H2O2 using the Karnovsky-Roots staining reaction product as a catalyst. Mounted longitudinal cryosections of mouse sternocleidomastoid muscle were fixed and stained in one step on the slide with paraformaldehyde plus the Karnovsky-Roots stain, followed by DAB-H2O2 secondary staining. The tissues were then processed for in situ hybridization and probed for the acetylcholine receptor (AChR) epsilon-subunit mRNA, known to be localized at the NMJ. The probe was labeled with 33P, which is ideal for in situ hybridization. By this procedure, the endplate stain was retained even after the hybridization and autoradiographic procedures, and the developed grains due to radiolabeling of the AChR epsilon-subunit mRNA were localized at readily identified endplates.
Identification of gastric H,K-ATPase in an early vertebrate, the Atlantic stingray Dasyatis sabina.
Authors: Smolka A J , Lacy E R , Luciano L , Reale E
Published: April 2011

Virtually all vertebrates acidify their gastric contents to a pH between 0.8 and 2.0. In mammals, acid secretion is mediated by a K-stimulated proton-translocating adenosine triphosphatase (H,K-ATPase), which establishes a million-fold gradient of protons across the apical membrane of the gastric parietal cell. The earliest phylogenetic appearance of gastric acid secretion is in cartilaginous fish, and we sought to verify in this class (Chondrichthyes) the presence and distribution of H,K-ATPase in gastric epithelial cells. An antibody against a synthetic peptide based on the C-terminus of pig H,K-ATPase alpha-subunit was localized in the gastric glands of the Atlantic stingray Dasyatis sabina. The C-terminal antibody stained all cells with tubulovesicles and the apical membrane domain of mucous neck cells. In proximal stomach, gastric glands showed the strongest immunoreactivity in cells close to the isthmus; in the distal stomach, strongest immunoreactivity was found in cells at the base of the glands. Oxyntic cells were more intensely immunoreactive than oxynticopeptic cells. This antibody labeled a single band of M(r) 100,600 on immunoblots of D. sabina gastric microsomes. These results show the earliest phylogenetic occurrence of a gastric ATPase in putative acid-secreting cells and suggest that this enzyme shares structural features with mammalian H,K-ATPase.
Ultrastructural localization of E-cadherin cell adhesion molecule on the cytoplasmic membrane of keratinocytes in vivo and in vitro.
Authors: Horiguchi Y , Furukawa F , Fujita M , Imamura S
Published: April 2011

We examined the ultrastructural localization of E (epithelial)-cadherin cell adhesion molecules by immunoperoxidase electron microscopy on the epithelium of mouse intestine, epidermis of human skin, and cultured human keratinocytes. The in vivo studies demonstrated that E-cadherin was present at the intermediate junction but not at the desmosome of the mouse intestinal single epithelium, and was found on the cytoplasmic membranes of keratinocytes with condensation in the intercellular space of the desmosomes, except for the basal surface of the basal cells. In vitro studies demonstrated that keratinocytes cultured in medium containing a low Ca2+ concentration (0.1 mM) lacked the tight connection through desmosomes, and that E-cadherin showed diffuse distribution and dot-like accumulation around the free surface of the cytoplasmic membrane. In culture medium containing a high concentration of Ca2+ (0.6 mM), keratinocytes formed desmosomal adhesion structures in which E-cadherin was accumulated. The free surface of the keratinocytes in this medium showed weaker distribution and a lesser amount of dot-like accumulation of E-cadherin than that in a low Ca2+ condition. These findings suggest that the distribution pattern of the E-cadherin cell adhesion molecules on the keratinocytes is different from that on the single epithelium of the intestine, and that E-cadherin on the cytoplasmic membrane of the keratinocytes shifts to the desmosomes under physiological conditions, participating in adhesion in association with other desmosomal cadherins.
Immunocytochemical localization of endogenous anti-thrombin III in the vasculature of rat tissues reveals locations of anticoagulantly active heparan sulfate proteoglycans.
Authors: Xu Y , Slayter H S
Published: April 2011

We localized endogenous anti-thrombin III (ATIII) by light and electron microscopic immunocytochemical staining in cryostat and ultra-thin frozen sections of 10 different rat tissues, using rabbit alpha-human ATIII antibody that was shown to crossreact strongly with rat ATIII. EM immunocytochemical methods revealed discrete deposits of endogenous ATIII (absent after heparinase treatment), and thus by inference anticoagulantly active heparan sulfate proteoglycans (HSPGs) at a resolution of 10-20 nm, or an order of magnitude better than autoradiography or LM. ATIII was found in variable amounts almost entirely in the subendothelial space of blood vessels in various rat tissues. In kidney, ATIII was found immediately beneath the endothelium, in concentrated clusters associated with the vascular basement membrane. Equally important is the observed variation in expression of ATIII in the various tissues studied (i.e., kidney > liver, aorta, lung, spleen, adrenal > intestine, muscle, brain). On the basis of these observations, we confirm a model in which vascular abluminal and, perhaps to a much smaller extent, luminal anticoagulantly active HSPGs regulate coagulation mechanism activity, either by serving as a reserve of anticoagulant or by modulating the ambient function of the coagulation cascade.
A novel nickel avidin-biotin-peroxidase method for histochemical visualization of neurofibrillary tangles, senile plaques, and neuropil threads.
Authors: Cullen K M
Published: April 2011

A reliable new method was developed for detecting neurofibrillary tangles, senile plaques, and neuropil threads in human neural tissue. Excellent morphological definition of the pathological structures was achieved with this procedure without staining normal neuronal and glial elements. The technique was applied to cortical tissue from eight patients with Alzheimer's disease and three controls. Histological sections from these cases were incubated in an avidin-biotin-peroxidase complex solution containing 0.5% nickel ammonium sulfate, followed by visualization in 3,3'-diaminobenzidine and H2O2. Although the avidin-biotin system is routinely used in immunohistochemistry, no antibodies were employed in the present procedure. This technique has advantages over silver impregnation methods because it requires very little monitoring of critical steps and yields superior results. The method is suitable for processing large numbers of tissue sections per staining run, and the results are highly reproducible. These features are advantageous in research studies comparing the distribution of lesions in large numbers of cases. The precise mechanism of staining has not been determined; however, conditions such as nickel concentrations, pH, and avidin-biotin-peroxidase complex concentrations were varied to examine critical steps in the process.
Ultrastructural localization of major basic protein in the human eosinophil lineage in vitro.
Authors: Dvorak A M , Furitsu T , Estrella P , Letourneau L , Ishizaka T , Ackerman S J
Published: April 2011

We examined the ultrastructural localization of (a) a secondary granule matrix protein--eosinophil peroxidase (EPO)--by cytochemistry, (b) a secondary granule core protein (major basic protein, MBP) by immunogold labeling, and (c) a primary granule protein (the Charcot-Leyden crystal protein, CLC protein) by immunogold labeling in eosinophilic myelocytes (EMs) and mature, activated eosinophils that differentiated from umbilical cord blood progenitors cultured in the presence of recombinant human interleukin-5 (rhIL-5). These studies provide the first substructural localization of MBP to condensing cores of immature secondary granules of EMs, as well as identification of unicompartmental, MBP-rich secondary granules that are devoid of matrix compartments and EPO content and are not primary granules by virtue of their lack of CLC protein. These granules occur in quantity in IL-5-activated mature human eosinophils, which have previously been shown to actively transport EPO from the matrix compartments of their secondary granules to the extracellular milieu in smooth membrane-bound cytoplasmic vesicles, a secretory process termed piecemeal degranulation, whereby eosinophils progressively empty cytoplasmic granules of their contents in the absence of classical granule extrusion.
Distinct intracellular lysozyme content in normal granulocytes and monocytes: a quantitative immunoperoxidase and ultrastructural immunogold study.
Authors: Resnitzky P , Shaft D , Yaari A , Nir E
Published: April 2011

Using two different immunological methods, we performed a quantitative estimation of lysozyme (LZ) in normal mature granulocytes and monocytes. An immunoperoxidase reaction for LZ in granulocytes and monocytes of 10 healthy donors measured by a scanning microdensitometer as arbitrary units showed a significantly higher LZ content in granulocytes than in monocytes. An ultrastructural immunogold reaction (IGR) for LZ performed on post-embedded thin sections showed a higher number of total gold grains in neutrophilic granulocytes than in monocytes. In monocytic granules we found a high density of gold grains per micron 2, whereas in granulocytic granules lower values were obtained. In granulocytes, LZ was found in both primary myeloperoxidase (MPO)-positive and secondary MPO-negative granules, and in monocytes the granules showed weak MPO reactivity and high LZ content. Granulocytes previously subjected to phagocytosis of bacteria and latex particles showed release of LZ on degranulation inside the phagosome, whereas in monocytes the granules remained outside the phagosome and released LZ without degranulation. Our study demonstrated a significantly higher total LZ content in granulocytes, a higher granular LZ content in monocytes, and release of LZ from intact monocyte granules during phagocytosis.
In vivo demonstration of subcellular localization of anti-transferrin receptor monoclonal antibody-colloidal gold conjugate in brain capillary endothelium.
Authors: Bickel U , Kang Y S , Yoshikawa T , Pardridge W M
Published: April 2011

To study transcytosis at the blood-brain barrier (BBB) in vivo, we used the internal carotid artery perfusion technique in rats. Brain uptake of the OX26 anti-transferrin receptor antibody (IgG2a) coupled to 5-nm colloidal gold (OX26-Au) was morphologically examined after infusion or perfusion into the carotid artery for 10 min. The brain tissue was subsequently perfusion-fixed with 2% glutaraldehyde. By light microscopy, silver enhancement of vibratome sections revealed staining of the vascular tree. Electron microscopy showed binding of gold particles at the luminal plasma membrane of brain capillary endothelia, endocytosis in vesicles (50-100 nm), and particles beyond the abluminal plasma membrane. Studies were performed with an IgG2a isotype control, UPC10. Internal carotid artery infusion of [125I]-UPC10 showed no evidence of brain uptake or binding to endothelium. However, microvessel structures were identified after silver enhancement of vibratome sections of brain following internal carotid artery infusion of 5-nm colloidal gold conjugates of UPC10 (UPC10-Au). The morphologically observed binding of UPC10-Au to brain microvessels may be induced by gold conjugation. These studies describe the use of gold conjugates of antibodies to delineate the subcellular pathway involved in transcytosis through the BBB.
Modified indirect immunodetection allows study of murine tissue with mouse monoclonal antibodies.
Authors: Hierck B P , Iperen L V , Gittenberger-De Groot A C , Poelmann R E
Published: April 2011

The indirect immunodetection method is powerful in detecting antigens in situ, but to date mouse monoclonal antibodies (MAbs) could not be used in immunohistochemical studies of murine tissues without severe background staining. We report here a modification of this method in which mouse MAbs are used to detect murine antigens in cryosections. Before application to the section, mouse MAbs and conjugated anti-mouse antiserum were allowed to complex in vitro. After blocking of the unbound secondary antiserum with normal mouse serum, standard immunohistochemistry was performed. Fifty percent of a randomly chosen panel of over 40 mouse MAbs recognized their antigens in our model system. Adaptation of, for example, the fixation protocol can probably even increase this number. An MAb to the intermediate filament protein desmin, staining both smooth and striated muscle, was used to demonstrate this technique in cryosections of 15-day-old mouse embryos. In contrast to standard immunohistochemistry with the same antibodies under the same conditions, background staining was completely absent with this technique. With this modification to the well-established indirect detection method, the usefulness of mouse MAbs is significantly increased.
Microwave-stimulated antigen retrieval is pH and temperature dependent.
Authors: Evers P , Uylings H B
Published: April 2011

The effects of the pH of solutions used in microwave-stimulated antigen retrieval were investigated for two antibodies commonly used in neurosciences, MAP-2 and SMI-32. A prolonged fixation time (longer than 1 week) prevents good immunohistochemical staining of these and many other antigens in paraffin and vibratome sections. On human brain tissue stored for 17 months in formaldehyde, the effects of different solutions during microwave treatment were compared, i.e., 4% aluminum chloride (pH 2.75), 0.1 N hydrochloric acid (pH 0.98), and citrate buffers (pH 2.5, 4.5, 6.0), together with the influence of temperature and microwave irradiation. We conclude that the pH and temperature of the solutions and the irradiation time are of great importance for the retrieval of antigens, but in a different way for the two antibodies studied. The SMI-32 staining results are optimal with citrate buffer at pH 2.5 and 2-hr irradiation at 90 degrees C, whereas MAP-2 staining results are optimal with citrate buffer at pH 4.5 and 10-min full-power irradiation. In the other conditions the staining is less intensive or less homogeneously distributed over the section.
Quantitation of P450 aromatase immunoreactivity in human ovary during the menstrual cycle: relationship between the enzyme activity and immunointensity.
Authors: Suzuki T , Sasano H , Sasaki H , Fukaya T , Nagura H
Published: April 2011

To understand changes associated with the menstrual cycle in the human ovary, it is very important to examine chronological changes in P450 aromatase (P450arom) enzymatic activity in the normal cycling ovary. Therefore, we initially examined the correlation between intensity of P450arom immunoreactivity and its biochemical enzymatic activity in five estrogen-producing human cancer cell lines (HHUA, Ishikawa, HEC-59, OMC-2, and MCF-7). P450arom immunointensity per cell was evaluated by the CAS 200 computed image analysis system, and its catalytic activity per 10(6) culture cells was analyzed by the tritiated water method. A significant correlation (r = 0.959) was demonstrated between P450arom immunoreactivity and enzymatic activity under optimal conditions of tissue fixation and immunohistochemical procedures. We then investigated P450arom immunointensity in 31 specimens of normal cycling human ovaries to examine chronological changes in P450arom activity per cell throughout the menstrual cycle. In the follicular phase, P450arom was observed in the granulosa cells of one selected antral follicle per case during the mid- to late proliferative period, and its immunointensity per granulosa cell in the follicle was not significantly different between mid- and late proliferative periods, although serum estradiol level was markedly elevated in the late proliferative period. In the luteal phase, both P450arom immunointensity per luteinized granulosa cell in a corpus luteum and serum estradiol level reached a peak in the mid-secretory period. These findings indicate that different factors may influence ovarian P450arom activity during the follicular and luteal phases, i.e., an increased number of granulosa cells in the selected follicle during the follicular phase but changes in P450arom activity per luteinized granulosa cell in the corpus luteum during the luteal phase.
Localization of sugar-binding sites in Staphylococcus aureus using gold-labeled neoglycoprotein.
Authors: Morioka H , Suganuma A , Tachibana M
Published: April 2011

We studied post- and pre-embedding staining of sugar-binding sites on thin sections of Staphylococcus aureus with an electron microscopic neoglycoprotein-gold technique. Although gold particles of cellobiosyl bovine serum albumin (BSA)-glycosylated BSA-, lactosyl BSA-, and melibiosyl BSA-gold did not label, heavy labeling of N-acetylglucosaminide-BSA-gold was observed in both the cell wall and the cytoplasm on Spurr-embedded thin sections of S. aureus. Inhibition of labeling with wheat germ agglutinin-biotin and N-acetylglucosaminidase indicated that the labeling was due to N-acetylglucosamine. These data suggested that molecules that bind specifically with N-acetylglucosamine occur in the cell wall and cytoplasm of S. aureus. Pre-embedding staining revealed that these molecules are abundant at the surface of the cell wall and that the abundance differs depending on the bacterial strain. An N-acetylglucosamine-specific lectin-like substance, glucosaminidase, and toxins are proposed as candidates for molecules responsible for the labeling, and the possible functional significance of the findings is discussed briefly.
Analysis of chick somite myogenesis by in situ confocal microscopy of desmin expression.
Authors: Borman W H , Yorde D E
Published: April 2011

We explored the relationship in chick embryos between somitogenesis and the onset of somite myogenesis by immunodetection of the muscle-specific intermediate filament protein desmin. Early somite desmin expression was detected by whole-mount in situ confocal microscopy. No detectable somite desmin was observed in embryos of 15 somites (Stage 12) or younger. In embryos having between 16 and 26 somites (Stages 12-15), desmin could be detected in somites positioned increasingly more caudal in the embryo. Finally, in embryos of 27 somites (Stage 16) and older, somite desmin expression was consistently present in all but the caudal-most six somites. Although the rate of somite formation is fairly constant, the rate of observed somite desmin expression progressing caudally in the embryo is greater initially than the rate of segmentation. After an embryo has formed about 27 somites, the rate of desmin appearance parallels the rate of segmentation at a distance of about six somites. This result suggests that very early somite myogenesis is not linked to somitogenesis.
Non-isotopic in situ hybridization method for mitochondria in oncocytes.
Authors: Varma V A , Cerjan C M , Abbott K L , Hunter S B
Published: April 2011

We used in situ hybridization to specifically identify mitochondria in a series of formalin-fixed, paraffin-embedded oncocytic lesions. Digoxigenin-labeled DNA probes were generated by the polymerase chain reaction (PCR), with primers designed to amplify a mitochondrion-specific 154 BP sequence within the ND4 coding region. Probes were hybridized with mitochondrial DNA under stringent conditions. Oncocytes were strongly and consistently stained, reflecting the high copy number of mitochondrial DNA within these cells. Because of the presence of endogenous biotin within mitochondria, digoxigenin is preferable to biotin as a label for detection of mitochondria.
Double epi-illumination microscopy with separate visualization of two antigens: a combination of epi-polarization for immunogold-silver staining and epi-fluorescence for alkaline phosphatase staining.
Authors: van der Loos C M , Becker A E
Published: April 2011

We present a method for an epi-illumination immunohistochemical double staining approach. The method combines the use of an immuno-alkaline phosphatase technique and the immunogold-silver technique, visualized with epifluorescence and epi-polarization illumination, respectively. Out of six tested alkaline phosphatase activity-revealing methods, only the reaction product obtained with the Becton Dickinson CAS Red kit showed an intense red fluorescence with a rhodamine filter set and no signal with epi-polarization illumination. The silver precipitate did not exhibit any signal with the rhodamine filter set. This allows separate observation and photographic recording of two antigens in one tissue section, an objective that cannot be achieved with conventional immunoenzyme double staining methods. The double epi-illumination approach presented is compatible with different immunoenzyme double staining protocols.
F(ab) secondary antibodies: a general method for double immunolabeling with primary antisera from the same species. Efficiency control by chemiluminescence.
Authors: Negoescu A , Labat-Moleur F , Lorimier P , Lamarcq L , Guillermet C , Chambaz E , Brambilla E
Published: April 2011

We propose a general solution to the problem of using antibodies originating in the same species for double immunohistochemical labeling. It relies on the use of a two-step protocol in which a secondary polyclonal monovalent F(ab) antibody present in the first step blocks access in the second of the secondary antibody to the primary antibody, which is continuously present from the first step. The monovalence of the F(ab) fragment eliminates the possibility of its linking the primary antibody from the second step. We designed two efficiency tests to explore the limits of the method by the very sensitive chemiluminescent system applied to sections of human pituitary tissue. They confirmed both the validity of the method and the necessity of adapting working conditions to obtain a complete lack of interference.
Localization of EGF receptors in frozen tissue sections by antibody and biotinylated EGF-based techniques.
Authors: Reeves J R , Cooke T G , Fenton-Lee D , McNicol A M , Ozanne B W , Richards R C , Walsh A
Published: April 2011

We developed a sensitive EGF receptor detection method for frozen tissue sections using biotinylated EGF as the primary reagent. The method was directly compared with an immunohistochemical technique based on an anti-EGF receptor monoclonal antibody (MAb EGFR1) in normal human and rat tissues and in human tumors. The method was more sensitive than a previously published biotinylated EGF-based technique. In normal human tissues and in 37 of the 50 tumors, the binding pattern mirrored that of positive staining with EGFR1. Five further tumors showed weak immunoreactivity, but in these no binding of biotinylated EGF was detected. The remaining eight tumors were negative by both techniques. The discordant cases may reflect a lower level of sensitivity of the ligand-binding technique or, alternatively, abnormal receptors may have been expressed in these tissues. EGF receptors could be detected in rat liver with biotinylated EGF but not with the antibody, indicating the usefulness of the ligand-based technique in cross-species studies.
Ultrastructural immunogold localization of osteopontin in human gallbladder epithelial cells.
Authors: Qu-Hong  , Brown L F , Senger D R , Geng L L , Dvorak H F , Dvorak A M
Published: April 2011

We used a post-embedding ultrastructural immunogold method to localize osteopontin in human gallbladder epithelial cells. This glycoprotein, originally described in bone but recently found to have a much wider distribution in many epithelia and in some mesenchymal cells, was present in the filamentous glycocalyx, small apical cytoplasmic smooth membrane-bound vesicles, large membrane-bound cytoplasmic granules, and in portions of the Golgi complex in gallbladder columnar epithelial cells. These findings suggest that newly synthesized osteopontin is packaged in Golgi-derived granules that release their contents by classical exocytosis from the cell surface. At least a portion of secreted osteopontin remains on the cell surface, where it becomes integrated into the filamentous glycocalyx coating the luminal surface of gallbladder epithelial cells.
Immunogold electron microscopic localization of timothy grass (Phleum pratense) pollen major allergens Phl p I and Phl p V after anhydrous fixation in acrolein vapor.
Authors: Grote M , Dolecek C , Van Ree R , Valenta R
Published: April 2011

We used the vapor phase of acrolein as an anhydrous fixative for timothy grass pollen in an immunogold double-labeling localization study of two different major allergens, Phl p I and Phl p V. More than 48 hr of fixation were needed for the subcellular pollen structures to be satisfactorily stabilized. The immunoreactivity of acrolein-fixed pollen allergens was not destroyed even after prolonged acrolein fixation. By immunoblotting, the two allergens differ in their immunological and structural characteristics. Electron microscopic localization traced the allergens at least partially to different subcellular pollen compartments.
Assessment of the labeling index of cohorts of the anterior pituitary cell population in phenobarbital-treated male rats by a double immunohistochemical technique for bromodeoxyuridine and pituitary hormones.
Authors: Jones H B , Harbottle S J , Bowdler A L
Published: April 2011

A previous study demonstrated that administration of phenobarbital for up to 7 days to male AP Wistar rats caused alterations in labeling indices (LIs) of several different tissues as determined by immunohistochemical visualization of bromodeoxyuridine (BrdU) incorporation into S-phase nuclei. The pivotal role of the pituitary gland in the function of the endocrine system and changes in circulating hormone levels that result from administration of xenobiotics prompted our consideration of the possible changes in LIs of individual cohorts of the anterior pituitary cell population that may occur as a specific functional adaptation during phenobarbital administration. We evaluated the LIs of individual anterior pituitary cell cohorts by modifying a double immunohistochemical staining method for bromodeoxyuridine and pituitary hormones using a sequential peroxidase-anti-peroxidase (PAP)/alkaline phosphatase-anti-alkaline phosphatase (APAAP) method employing diaminobenzidine and New Fuchsin chromogens, respectively. The method was robust and reproducible. Differences were noted in individual anterior pituitary cohort LIs between control and phenobarbital-treated groups, although no statistically significant difference was evident. We conclude that no detectable effects on individual cohort LIs were induced by treatment with phenobarbital for up to 7 days and that any functional adaptation to treatment was associated with increased hormone release. We believe that the visualization, identification, and quantitation of replicating cells in specific hormone-positive cohorts of the anterior pituitary cell population provide opportunities for understanding the influence of xenobiotics and disease processes on pituitary function.
Fluorescence overlay antigen mapping of the epidermal basement membrane zone: I. Geometric errors.
Authors: Bruins S , de Jong M C , Heeres K , Wilkinson M H , Jonkman M F , van der Meer J B
Published: April 2011

To identify in tissue sections the relative positions of antigen distributions close to the resolving power of the microscope, we have developed the fluorescence overlay antigen mapping (FOAM) procedure. As this technique makes high demands on the geometric fidelity of the overlay image, it is essential to recognize geometric errors resulting from optical imperfections. This applies in particular to the image shift difference (ISD) that may routinely occur during fluorescence overlay. We describe here procedures for assessment and mechanical correction of the ISD in tissue sections. Furthermore, we describe an alignment verification test to assess the accuracy of the ISD correction procedure, using collagen Type VII as the geometric verification marker. These procedures should enable reliable evaluation of relative antigen distributions in tissue sections using photomicrographic multicolor fluorescence overlay. Further details of the FOAM technique, such as color fidelity and its utility for diagnostic and research purposes, will be published separately.
NADPH-diaphorase activity of nitric oxide synthase in the olfactory bulb: co-factor specificity and characterization regarding the interrelation to NO formation.
Authors: Spessert R , Wohlgemuth C , Reuss S , Layes E
Published: April 2011

The neuronal form of the enzyme nitric oxide synthase (nNOS) synthesizes the messenger molecule nitric oxide (NO). In addition to NO formation, nNOS exhibits a so-called NADPH-diaphorase (NADPH-d) activity. This study focused on the characterization of NADPH-d activity with regard to NO formation in the rat olfactory bulb. In this area of the brain pronounced staining is localized in discrete populations of neuronal somata and in olfactory glomeruli. Diaphorase staining combined with demonstration of nNOS by polyclonal antibodies revealed that NADPH-d activity of neuron somata is associated with nNOS immunoreactivity. It is concluded that neuron somata exhibit NADPH-d activity of nNOS. NADPH-d activity of nNOS did not utilize beta-NADH or alpha-NADPH. Moreover, NADPH-d activity was inhibited in the presence of alpha-NADPH. Dichlorophenolindophenol (DPIP), an artificial electron acceptor and an inhibitor of NO formation, totally suppressed NADPH-d staining of neurons, supporting the concept that the NADPH-d of neuron somata is due to nNOS. Cytochrome C, miconazole, EGTA, and trifluoperazine, which have been reported to inhibit cytochrome P450 reductase activity of NOS, did not affect NADPH-d staining. Hence, NADPH-d activity of NOS does not involve cytochrome P450 reductase activity as required for NO formation. Contrary to NADPH-d activity of neuron somata, staining of olfactory glomeruli was not co-localized with nNOS immunoreactivity. Glomerular staining was also observed in the presence of beta-NADH and alpha-NADPH. Further, it was unchanged in the presence of the NO formation inhibitor DPIP. Hence, the glomerular staining in the presence of NADPH is not due to the NADPH-d activity of NOS. We conclude that staining of neuronal structures in the presence of NADPH does not necessarily represent NADPH-d activity of NOS.
Application of electron microscopic immunocytochemistry to the human kidney: distribution of type IV and type VI collagen in normal human kidney.
Authors: Zhu D , Kim Y , Steffes M W , Groppoli T J , Butkowski R J , Mauer S M
Published: April 2011

We used immunogold electron microscopic (IEM) techniques with periodate-lysine-paraformaldehyde-fixed and Lowicryl-embedded or cryopreserved tissues to study the distribution of alpha 1(IV) and alpha 3(IV) chains of Types IV and VI collagen in glomerular basement membrane (GBM) and mesangial matrix of glomeruli in normal human kidneys. Monoclonal antibodies to alpha 1(IV) and alpha 3(IV) collagen chains and Type VI collagen could be detected only with cryoultramicrotomy, whereas polyclonal anti-Type IV collagen antibody was detectable in Lowicryl-embedded tissue. Ultrastructural detail was better preserved in the Lowicryl-embedded tissue. IEM labeling provided more detailed information as to the site-specific array of these extracellular matrix molecules in glomeruli than did immunofluorescent microscopy. The labeling of alpha 1(IV) collagen chain was distributed mainly along the endothelial side of glomerular basement membrane and the mesangial matrix. Mesangial GBM was relatively poorly labeled compared with that of mesangial matrix. In contrast, the alpha 3(IV) chain was detected throughout the thickness of the GBM, but there was no labeling of mesangial matrix. Type VI collagen distribution was identical to that of the alpha 1(IV) chain within the glomerulus but was also associated with interstitial collagen fibrils. This study documents and details the heterogeneous distribution of Type IV and VI collagen chains within the normal human glomerulus and provides the framework for the study of these matrix components in human glomerular diseases.
Immunohistochemical detection of ornithine decarboxylase in individual cells: potential application for in vitro chemosensitivity assays.
Authors: Shayovits A , Bachrach U
Published: April 2011

Ornithine decarboxylase (ODC), which catalyzes the rate-limiting step in the biosynthesis of polyamines, can serve as a marker of proliferation. The presence of ODC protein in individual cells was quantitatively detected by an immunofluorescence assay using an ACAS 570 computerized fluorescence microscope. ODC was detected in KB-3-1 human epithelial carcinoma cells grown in the absence of any drug. Vinblastine, which inhibits cell growth, caused the disappearance of ODC. On the other hand, ODC was detected in multidrug-resistant cells grown in the absence or in the presence of vinblastine. NIH 3T3 fibroblasts transformed by the c-Ha-ras oncogene also contained ODC protein. This protein disappeared when the cells were treated with cycloheximide, which inhibits cell proliferation. These findings suggest that ODC can be detected in individual cells by immunofluorescence. Whether this method can be used for in vitro chemosensitivity tests remains to be studied.
A multilabeling technique for simultaneous demonstration and quantitation of Ki-67 and nucleolar organizer regions (AgNORs) in paraffin-embedded tissue.
Authors: Munakata S , Hendricks J B
Published: April 2011

Although many investigators have demonstrated a relationship between argyrophilic nucleolar organizer regions (AgNORs) and Ki-67 expression in solid tumors, no previous studies have simultaneously assessed the relationship between AgNOR and Ki-67 expression in paraffin-embedded tissue. We describe a method for simultaneous demonstration and quantitation of Ki-67 and AgNORs in routinely processed tissue. The Ki-67 equivalent monoclonal antibody MIB1, which can detect proliferative activity in routinely processed tissue with microwave heating, was employed. Fresh human tonsil tissue was fixed in formalin and embedded in paraffin for Ki-67/AgNOR dual staining. Image analysis was employed for quantitation of AgNOR staining in Ki-67-positive and Ki-67-negative nuclei. The double-staining procedure had no measurable effect on the individual parameters: Ki-67 labeling index, mean AgNOR number (NN), and NOR percentage nuclear area (NPNA). However, microwave processing for Ki-67 immunostaining significantly increased nuclear area (NA) and AgNOR area (AA). A significant difference was found between Ki-67-positive and Ki-67-negative cells for NN (p < 0.001), NA (p < 0.001), AA (p < 0.001), and NPNA (p < 0.001). These results suggest a direct relationship between AgNOR and Ki-67 in paraffin-embedded tissue.
Application of fine-needle aspiration to the demonstration of ERBB2 and MYC expression by in situ hybridization in breast carcinoma.
Authors: Soini Y , Mannermaa A , Winqvist R , Kamel D , Poikonen K , Kiviniemi H , Pääkkö P
Published: April 2011

Thirteen consecutive fine-needle aspirates of breast carcinoma and five selected breast tumor cell lines were analyzed for ERBB2 and MYC mRNA expression by in situ hybridization. To compare the level of mRNA synthesis with those of gene amplification and oncoprotein synthesis, all tumors were also analyzed by Southern blot analysis, and for ERBB2 also by immunohistochemistry. Expression of ERBB2 mRNA was observed in eight tumors. MYC expression was observed in all tumors studied. Three tumor cell lines expressed both ERBB2 and MYC (SK-BR-3, HeLa, HT-29) and two only MYC (SK-LU-1, HL-60). Only one tumor showed amplification of ERBB2 and two of MYC. In all three cases there was a considerable increase in corresponding mRNA synthesis as detected by in situ hybridization. By immunohistochemistry, four cases showed either patchy areas or uniformly distributed, membrane-bound ERBB2 immunoreactivity. All except one case showed increased ERBB2 mRNA synthesis. There was a clear association between the quantity of ERBB2 mRNA and oncoprotein expression. The results show that in situ hybridization of fine-needle aspiration material is a sensitive method to detect increased expression of the ERBB2 and MYC oncogenes in breast carcinoma. Furthermore, this study indicates that in a majority of cases some other mechanism that gene amplification appears responsible for the increased gene expression. It is also possible that Southern blot analysis is not a sensitive enough method to detect gene amplifications in the heterogeneous breast tumors, which usually also contain stromal tissue. The fact that not all cases with elevated ERBB2 mRNA synthesis were immunohistochemically positive suggests that either immunohistochemistry (after fixation with 10% formalin) is a less sensitive method than in situ hybridization to detect abnormal gene expression or that there are cases in which the oncoprotein synthesis is for some reason depressed, even though there is an increase in gene transcription.
A Paneth cell analogue in Xenopus small intestine expresses antimicrobial peptide genes: conservation of an intestinal host-defense system.
Authors: Reilly D S , Tomassini N , Bevins C L , Zasloff M
Published: April 2011

Antimicrobial peptides are a widespread component of host defense. We characterized the tissue distribution and cellular localization of expression of the magainin family of antimicrobial peptide genes in Xenopus laevis. Two genes from this family, magainin and PGLa, are expressed at high levels in the skin and throughout the gastrointestinal tract. Magainin and PGLa mRNAs are synthesized in the granular multinucleated cell (GMC) of the gastric mucosa, a cell shown previously to contain magainin and PGLa peptides by immunohistochemical methods. In addition, we have localized magainin and PGLa mRNAs to distinct cells of Xenopus small intestine. Further characterization of this large, granule-filled cell by electron microscopy demonstrates features in common with the Paneth cell of mammalian small intestine, previously identified as a site of expression of antimicrobial peptide genes of the defensin family in mouse and human. Our identification of granule-laden, eosinophilic intestinal cells in Xenopus as a site of magainin and PGLa antimicrobial peptide gene expression suggests that these cells are functional analogues of mammalian Paneth cells and further supports a conserved role of antimicrobial peptides in host defense of the vertebrate small intestine.
Expression of the class I alcohol dehydrogenase gene in developing rat fetuses.
Authors: Tietjen T G , Mjaatvedt C H , Yang V W
Published: April 2011

Class I alcohol dehydrogenase (ADH) is the principal enzyme responsible for ethanol oxidation in mammals. Although primarily regarded as an enzyme that functions in the adult, Class I ADH has been reported to be present in fetal tissues. By in situ hybridization, we demonstrated the tissue localization of the Class I ADH transcript in developing rat fetuses between Days 15 (E15) and 18 (E18) of gestation. Abundant transcripts were present in epidermis, lung, and urinary bladder. In these tissues, the messages were localized primarily to the superficial layer of the epithelium and increased with development. The liver exhibited significant signals only in the E18 fetus, when parenchymal hepatocytes first appeared. The E15 and E16 small intestines, with their epithelium arranged in a stratified fashion, displayed signals in the submucosal mesenchymal layer. By E17, a rearrangement of the intestinal epithelium into an almost monolayer configuration was observed. This change was associated with a redistribution of the ADH transcript to the surface of the epithelium. Further relocation of the messages was noted in the adult small intestine, in which they became concentrated in the base of the crypt. These findings indicate that expression of the rat class I ADH gene follows a dynamic course in specific epithelial tissues during fetal development. In addition, the apparent superficial localization of the ADH message in most of these tissues suggests that ADH functions in metabolizing either endogenously or exogenously derived alcohol substrates present in the fetal environment.
Preferential localization of heat shock protein 47 in dilated endoplasmic reticulum of chicken chondrocytes.
Authors: Kambe K , Yamamoto A , Yoshimori T , Hirayoshi K , Ogawa R , Tashiro Y
Published: April 2011

We investigated the distribution of heat shock protein 47 (hsp47) in cultured chicken embryonic chondrocytes and epiphyseal chondrocytes of tibial bones from 1-day-old to 6-week-old chickens. Northern blot and immunoblot analyses revealed that hsp47 exists in epiphyseal cartilage and cultured chondrocytes. Confocal laser immunofluorescence microscopy showed that hsp47 was localized mainly in the many granular structures found in the cytoplasm that contain Type II collagen. Epiphyseal cartilage and cultured chondrocytes were embedded in LR White resin and hsp47 was detected by protein A-immunogold electron microscopy. Gold particles were localized exclusively in the cisternal space of the endoplasmic reticulum (ER), and the labeling density of the cisternal space of the dilated ER was always higher than that of the non-dilated ER. In all the differentiating zones of epiphyseal cartilage, the labeling density was highest in the hypertrophic cells. These findings suggest that hsp47 plays an important role(s) in the synthesis, processing, and assembly of Type II collagen.