bluuebunny/crossref_metadata_2025 huggingface dataset:
Localization of the G-protein G(o) in exocrine glands.
Authors: Watson E L , Oliver C , D'Silva N , Belton C M
Published: April 2011

The GTP-binding protein G(o) was localized immunohistochemically in the rat parotid gland and in other exocrine glands with specific G(o) antibodies. Immunohistochemical studies revealed that affinity-purified G(o alpha) polyclonal antibody (GO/85) immunoreacted primarily with duct cells of the rat parotid gland; immunoreactivity was also noted in duct cells of the rat submandibular, mouse parotid, and mouse submandibular glands. Light labeling of rat parotid and submandibular gland acinar cells was also noted. G(o alpha) antiserum (9072) differing in specificity for epitopes within G(o alpha) produced similar results. This antiserum also immunoreacted with rat submandibular duct cell secretory granule membranes. In contrast, in rat and mouse pancreas G(o alpha) antibodies immunoreacted primarily with islet cells. Duct cells were negative but there was light labeling of rat pancreatic acinar cells. The apparent duct specificity of G(o alpha) staining was further verified by demonstrating that G(o alpha) antibodies immunoreacted with HSG-PA cells, a human transformed salivary duct cell line. Specificity in immunohistochemical labeling of HSG-PA cells was confirmed by Western blot analysis. The results demonstrate that G(o) appears to be selectively expressed in the duct cells of rat parotid gland and other salivary glands. The selective enrichment of G(o) in duct cells suggests that this G-protein plays an important role in duct cell physiology.
Evidence for paracrine somatostatinergic regulation of gastrin gene expression by double-staining cytochemistry and quantitation.
Authors: Larsson L I , Houggaard D M
Published: April 2011

Gastrin is produced by gastric endocrine cells and regulates acid secretion and mucosal cell proliferation. Somatostatin inhibits gastrin secretion, and exogenous somatostatin was recently reported to decrease gastrin gene transcription and to reduce gastrin mRNA stability. Somatostatin cells extend cytoplasmic processes that contact gastrin cells and other cells. Using quantitative in situ hybridization combined with immunocytochemistry, we demonstrate that gastrin cells in close contact (< or = 0.2 micron) with somatostatin cells express significantly lower levels of gastrin mRNA than other gastrin cells. Our data show that cells with high levels of gastrin mRNA and no detectable contacts predominate at the base of the glands. Hence, the results provide evidence that endogenous somatostatin regulates gastrin gene expression in a subpopulation of gastrin cells and point to regulatory heterogeneity of these cells. This represents the first direct microscopic evidence for paracrine gene regulation. The new quantitative double-staining approach may be generally useful for studies of cell interactions and cell sociology.
Molecular anatomy of a skin gland: histochemical and biochemical investigations on the mucous glands of Xenopus laevis.
Authors: Schumacher U , Adam E , Hauser F , Probst J C , Hoffmann W
Published: April 2011

The purpose of this study was to investigate the structure and chemical composition of the mucous skin gland of Xenopus laevis by combined morphological and biochemical techniques. Protein backbones of mucins were localized immunohistochemically in the gland with anti-peptide antibodies. Acid mucins were demonstrated by conventional histochemical techniques and their terminal carbohydrate residues were localized by lectin histochemistry. A close correlation between antibody and lectin binding of the same glycoproteins was achieved on Western blots from isolated skin gland mucins, indicating that the lectin-binding sites were due to defined mucin molecules. The cone cell, thought to be a degenerative cell in the past, contained mucin granules with an electron-dense core, strong PAS reactivity, a special lectin-binding pattern, and localization of integumentary mucins FIM-B.1 and FIM-C.1. These results indicate that cone cells are a distinct cell type, elaborating and releasing particular mucins, and that functional heterogeneity of mucus-producing cells exists in the mucous skin glands of X. laevis.
Histochemical detection of binding sites for human growth hormone using biotinylated ligand.
Authors: Bentham J , Aplin R , Norman M R
Published: April 2011

Recombinant human growth hormone was covalently linked to biotin via a six-carbon spacer arm. Biotinylation was confirmed by electrophoresis and mass spectrometry showed that approximately 50% of the hormone was monobiotinylated. The modified growth hormone (GH) was shown to bind to the GH receptor of IM9 human lymphoid cells with an affinity of 0.55 x 10(9) M-1. Bioactivity of biotinylated GH measured in the Nb2 bioassay was 53.9% that of unlabeled GH. GH binding sites on human IM9 cells were visualized histochemically with the biotinylated hormone, a technique that provides a means of identifying receptors for GH on target cells in vitro.
Adaptation of the reverse hemolytic plaque assay to electron microscopy: a study of the individual secretory activity in prolactin cell subpopulations.
Authors: Vila-Porcile E , Picart R , Tougard C
Published: April 2011

We used the reverse hemolytic plaque assay (RHPA), a method for detecting secretion by single cells, to demonstrate functional heterogeneity among prolactin (PRL) cells of rat anterior pituitary at the light microscopic (LM) level. We attempted to adapt RHPA for electron microscopy (EM) to define the relationships between fine structure and secretory activity in individual PRL cells. A major modification of the technique, intended to improve preservation of ultrastructure, was allowing the cells to recover after their enzymatic dispersion from the pituitary tissue, through a brief (3-4 hr) culture step before the assay. Adaptation for EM was achieved by the use of plastic slides for construction of specialized Cunningham chambers, permitting application of all the EM procedures (flat embedding, punching of selected areas) usually employed for cultured pituitary monolayers. Moreover, immunocytochemical pre- and post-embedding methods were also applied for cell identification and study of subcellular hormone distribution. Such a modified RHPA enabled us to analyze the ultrastructure of plaque-forming cells surrounded by their companion red blood cell ghosts. The first results with EM RHPA showed that under basal conditions a subpopulation of PRL cells containing small granules (150-200 nm) was actively secreting, whereas PRL cells with large (300-600 nm) and irregular granules appeared to be stimulated by thyrotropin-releasing hormone or KCl. The EM RHPA technique described here might receive more general application and could be utilized for study of many other secretory systems.
Ultrastructural visualization and neurochemical characterization of spinal projections of primary sensory afferents from the nipple: combined use of transganglionic transport of HRP-WGA and glutamate immunocytochemistry.
Authors: Rousselot P , Poulain D A , Theodosis D T
Published: April 2011

We used transganglionic transport of the neuronal tracer horseradish peroxidase coupled to wheat germ agglutinin (HRP-WGA) and post-embedding immunogold staining to determine the spinal projections and neurochemical identity of sensory afferents originating from a discrete cutaneous area. After SC injection of tracer into the nipple of lactating rats and reaction with tetramethylbenzidine stabilized with diaminobenzidene (TMB-DAB) or DAB and cobalt (TMB-DAB-Co), we found labeled terminals in the internal part of the first two layers of the dorsal horn where they formed axodendritic synapses and, at times, central elements of glomeruli, synaptic complexes believed to be involved in the integration of sensory messages. Immunogold staining of ultra-thin sections of tissue reacted with TMB-DAB-Co revealed that many mammary afferents contained glutamate as putative neurotransmitter. This combined approach thus offers the possibility of marking a limited set of primary afferents, after capture of tracer by sensory receptors of restricted peripheral areas, to visualize their projections at the spinal level and to determine their neurochemical nature with electron microscopy.
Expression of IL-6 mRNA in normal rat and human pituitaries and in human pituitary adenomas.
Authors: Velkeniers B , Vergani P , Trouillas J , D'Haens J , Hooghe R J , Hooghe-Peters E L
Published: April 2011

Cells expressing IL-6 mRNA were detected by in situ hybridization in normal pituitaries. In normal untreated rat pituitary the expression was very low. Within hours after IP administration of liposaccharide, IL-6 mRNA accumulated in the anterior lobe of the pituitary. Production of IL-6 was monitored after dissociation and culture of pituitary cells. High levels (8000 pg/ml) were recovered after 72 hr in culture. In normal human pituitaries, less than 1% of cells expressed IL-6 mRNA or IL-6 receptor mRNA (IL-6-R mRNA). In gonadotropinomas, prolactinomas, and non-functioning adenomas, only rare, scattered positive cells were found for either IL-6 or IL-6-R. In contrast, both genes were highly expressed in ACTH- and GH-secreting tumors at the junction of adenoma and infiltrating fibrous tissue and around blood vessels. The combined expression of IL-6 and IL-6-R suggests that IL-6 acts in an autocrine or in a paracrine way in ACTH and GH adenomas.
Monoclonal antibodies against the protein core and glycosaminoglycan side chain of glomerular basement membrane heparan sulfate proteoglycan: characterization and immunohistological application in human tissues.
Authors: van den Born J , van den Heuvel L P , Bakker M A , Veerkamp J H , Assmann K J , Berden J H
Published: April 2011

We raised monoclonal antibodies (MAb) against the core protein and the heparan sulfate (HS) side chain of heparan sulfate proteoglycan (HSPG) from glomerular basement membranes (GBM). Anti-HSPG-core MAb were obtained after immunization of mice with HSPG purified from human GBM and the anti-HS MAb after immunization of mice with HSPG from rat glomeruli, which crossreacted with human HS and GBM HSPG. The specificity of the MAb was demonstrated by ELISA studies, Western blotting, inhibition experiments, and indirect immunofluorescence (IF) on kidney cryostat sections pre-treated with glycosaminoglycan (GAG)-degrading enzymes. Indirect IF on normal human kidney tissue showed prominent GBM staining for both MAb, with variable staining of the other renal basement membranes (BMs). By indirect immunoelectron microscopy (IEM), most intense staining was observed at the endothelial side of the GBM for both MAb, although the staining patterns were not identical. Both MAb were used to localize HSPG in human tissues by indirect IF. They bound to antigens present in the BMs of most tissues examined, including those of epithelia and endothelia. Differences between both MAb were observed for BMs of muscle cells, since the anti-HSPG core protein MAb (JM-72) staining was negative, whereas the anti-HS MAb (JM-403) clearly stained these structures. Comparison of our staining patterns in human tissues with the distribution of other anti-BM HSPG antibodies suggests that there are at least two types of BM HSPG, which have common epitopes on the HS side chains recognized by JM-403.
Fixation conditions affect the intensity but not the pattern of NADPH-diaphorase staining as a marker for neuronal nitric oxide synthase in rat olfactory bulb.
Authors: Spessert R , Layes E
Published: April 2011

NADPH-diaphorase (NADPH-d) is commonly used as a histochemical marker for the neuronal form of the enzyme nitric oxide synthase (NOS). A recent biochemical study showed that in broken-cell preparations NADPH-d activity did not fully represent NOS and that NOS-unrelated NADPH-d activity was suppressed during fixation. Because it is unknown whether fixation also affects NOS-associated NADPH-d activity, we investigated the effects of various widely used fixatives on NADPH-d staining in relation to NOS immunoreactivity, obtained with polyclonal antibodies, in rat olfactory bulb. We found that the intensity of NADPH-d staining associated with NOS, as well as that unrelated to NOS, depends on fixation conditions. Addition of glutaraldehyde or lysine/sodium periodate to the fixative decreased intensity of NADPH-d staining. Fixative-dependence of NADPH-d staining was observed not only in the presence of the "normal" co-substrate beta-NADPH but also in the presence of the stereoisomer alpha-NADPH. Unlike the staining intensity, the staining pattern of NOS-associated as well as NOS-unrelated NADPH-d did not change after treatment with various fixatives. Our findings are of considerable practical significance because it has become clear that fixation conditions affect the sensitivity but not the selectively of the NADPH-d reaction as a marker for the presence of NOS.
Nitric oxide synthase and NADP-linked glucose-6-phosphate dehydrogenase are co-localized in brush cells of rat stomach and pancreas.
Authors: Kugler P , Höfer D , Mayer B , Drenckhahn D
Published: April 2011

The epithelia of the respiratory and gastrointestinal tract and their appendages contain a distinct population of disseminated epithelial cells called brush cells or caveolated cells. On the basis of their structure, it was suggested that brush cells might serve as chemo- or volume receptors that play a role in certain aspects of gastrointestinal and bronchopulmonary secretion or motility. In the present study we provide first clues to a possible function of this widespread epithelial cell type. Brush cells of the rat gastric cardia and major pancreatic duct display strong immunoreactivity for nitric oxide synthase (NOS) and also exhibit high activity of NADPH-diaphorase. This NADPH-oxidizing activity was previously shown to be mediated by a specific domain of the sequence of the NOS. NADPH, in turn, appears to be delivered by glucose-6-phosphate dehydrogenase, which we found in brush cells at particularly high levels. We conclude that brush cells of the stomach and pancreas may represent a specialized population of paracrine cells that use nitric oxide as a messenger molecule to control certain gastrointestinal functions.
A novel triple-color detection procedure for brightfield microscopy, combining in situ hybridization with immunocytochemistry.
Authors: Speel E J , Jansen M P , Ramaekers F C , Hopman A H
Published: April 2011

We describe a fast light microscopic procedure for the simultaneous enzyme cytochemical detection of three different DNA target sequences in contrasting colors in both interphase and metaphase cell preparations. Chromosome-specific DNA probes labeled with either biotin, digoxygenin, or fluorescein were hybridized as a mixture and detected clearly and accurately by precipitates of the horseradish peroxidase-diaminobenzidine (PO-DAB, brown color), alkaline phosphatase-Fast Red (APase-Fast Red, red color), or horseradish peroxidase-tetramethylbenzidine (PO-TMB, green color) reaction, respectively. The PO-TMB reaction product was stabilized effectively by the addition of sodium tungstate to the reaction mixture, thus making the PO-TMB reaction now generally applicable to in situ hybridization (ISH). To avoid mixing of the precipitates of the two PO reactions used in the triple-color ISH method, the first detected PO activity was always completely inactivated by a mild acid treatment before the second one was applied. Finally, the cell preparations were embedded in a thin protein layer cross-linked by formaldehyde to ensure permanent stabilization of the enzyme reaction products and optimal visualization of color contrast. The triple-color ISH detection procedure could be combined with beta-galactosidase-5-bromo-4-chloro-3-indolyl-beta- D-galactoside (beta-Gal-BCIG) immunocytochemistry (ICC), leading to the simultaneous localization of multiple DNA targets and a protein target in the same cell. The described procedure may therefore be a valuable tool in the areas of cytogenetics, cell biology, and molecular pathology.
The histochemical G6PDH reaction but not the LDH reaction with neotetrazolium is suitable for the oxygen sensitivity test to detect cancer cells.
Authors: Griffini P , Vigorelli E , Jonges G N , Van Noorden C J
Published: April 2011

We used the oxygen sensitivity of the histochemical reaction to detect glucose-6-phosphate dehydrogenase (G6PDH) activity based on neotetrazolium (NT) reduction to discriminate cancer cells from normal cells. Formazan generation was strongly reduced in normal but not in malignant cells when the incubation was performed in oxygen instead of nitrogen. Competition for reductive equivalents between NT and oxygen via superoxide dismutase (SOD) has been suggested. Since SOD activity is usually decreased in cancer cells, NT reduction would not be hampered in these cells. We tested this hypothesis by demonstrating NAD-dependent lactate dehydrogenase (LDH) activity instead of NADP-dependent G6PDH activity in normal rat liver and colon, in human colon carcinoma, and in experimentally induced metastases of colon carcinoma in rat livers. Reactions for both enzymes were determined cytophotometrically in an atmosphere of pure oxygen or nitrogen. G6PDH acted as described previously, showing distinct activity in cancer cells but strongly reduced activity in normal cells after incubation in oxygen, but this was not the case with LDH because formazan was also generated in normal tissue in oxygen. It appeared that after 5 min of incubation at 37 degrees C the residual activity of G6PDH in an atmosphere of oxygen compared with nitrogen was 0% in normal liver tissue and 15% in normal colon epithelium, whereas in colon carcinoma and in colon carcinoma metastasis in liver it was 48% and 33%, respectively. The residual activity of LDH in oxygen was 30% in normal female rat liver, 75% in normal male rat liver, and 38% in normal colon epithelium, whereas the residual activity in colon carcinoma and metastases in liver was 54% and 24%, respectively. These experiments clearly indicate that the oxygen sensitivity phenomenon is not solely an effect of competition for reducing equivalents between NT and oxygen via SOD, because NADPH generated by G6PDH and NADH generated by LDH have a similar redox potential. Apparently the system is more complex. The role of specifically NADPH-converting cellular systems such as NADPH-cytochrome P450 reductase was excluded because incubations in the presence of exogenous NADPH as substrate for these systems revealed oxygen sensitivity. Involvement of NADPH-dependent lipid peroxidation in the oxygen sensitivity test is discussed.
High-activity carbonic anhydrase isoenzyme (CA II) in human gallbladder epithelium.
Authors: Juvonen T , Parkkila S , Parkkila A K , Niemelä O , Lajunen L H , Kairaluoma M I , Perämäki P , Rajaniemi H
Published: April 2011

Acidification of bile is one of the factors that prevents calcium precipitation and thereby gallstone formation. Carbonic anhydrase II (CA II) has previously been shown to be one of the key factors in the human alimentary tract that regulates the acid-base balance. We demonstrated CA II expression in the human gallbladder epithelium using immunohistochemical techniques, elucidated the CA II content of the epithelium by digital image analysis of the immunohistochemically stained enzyme in samples from 16 patients undergoing cholecystectomy, and correlated the results with the calcium content of the gallstones. Nine patients had symptomatic gallstone disease and seven an acalculous, histologically normal gallbladder. The patients were classified into two groups on the basis of the calcium content of their gallstones: no gallstones or gallstones containing no calcium (Group 1) and gallstones with 2-87% calcium by weight (Group 2). The immunohistochemical techniques showed distinct epithelial CA II-positive staining in most of the gallbladder samples, but digital image analysis revealed distinct variations in staining intensity among them. The median staining intensity index was significantly higher in Group 1 (0.4463) than in Group 2 (0.2376; p = 0.0262). The results suggest that CA II is abundantly expressed in the normal gallbladder epithelium and that decreased expression may be associated with the formation of calcified gallstones. These findings are relevant to the pathogenesis of gallstone disease.
Localization of cyclic adenosine 3',5'-monophosphate-responsive element (CRE)-binding proteins by southwestern histochemistry.
Authors: Koji T , Komuta K , Nozawa M , Yamada S , Nakane P K
Published: April 2011

Regulation of gene transcription requires an interaction between specific segments of nuclear DNA and specific proteins. We describe a method to localize the specific DNA-binding proteins using haptenized double-stranded (ds) DNAs. To demonstrate this method, an oligodeoxynucleotide (oligo-DNA) with a consensus base sequence of cyclic adenosine monophosphate-responsive element (CRE) (TGACGTCA) with three TTA repeats at the 5' end was synthesized. Since the CRE sequence is palindromic, the oligo-DNA was allowed to self-anneal and form ds DNA with three TTA repeats at both ends. The CRE ds-oligo-DNA was irradiated with UV light to form haptenic thymine-thymine (T-T) dimers. The haptenized CRE ds-oligo-DNA reacted by Southwestern analysis with a distinct set of proteins, previously identified as CRE-binding proteins, ranging from 40-90 KD. When the haptenized CRE ds-oligo-DNA reacted with frozen sections fixed with 4% paraformaldehyde in PBS (pH 7.4) followed by enzyme immunohistochemical localization of the T-T dimers, nuclei of intestinal epithelial cells and brain cells were heavily stained. Nuclear staining was blocked when the sections were reacted with the haptenized CRE ds-oligo-DNA in the presence of an excess amount of non-haptenized CRE ds-oligo-DNA. This method, henceforth referred as Southwestern histochemistry, should be a useful tool to localize proteins that bind to a specific DNA sequence and regulate the transcriptional activity of genes.
Expression of c-kit and kit ligand proteins in normal human tissues.
Authors: Lammie A , Drobnjak M , Gerald W , Saad A , Cote R , Cordon-Cardo C
Published: April 2011

The c-kit receptor and its cognate ligand, KL, play a critical role in melanogenesis, gametogenesis, and hematopoiesis. Studies on the expression of c-kit and KL have been primarily focused on mouse development. We undertook the present study to characterize the pattern of expression of these molecules in normal adult human tissues. Using immunohistochemistry and consecutive tissue sections from the same block, we evaluated a variety of well-preserved normal tissues for c-kit and KL microanatomic distribution. c-kit protein was identified in tissue mast cells, melanocytes, glandular epithelial cells of breast, parotid, dermal sweat, and esophageal glands. Scattered c-kit immunoreactivity was also observed for testicular and ovarian interstitial cells. A striking regional distribution of c-kit was detected in the central nervous system, particularly in the cerebellum, hippocampus, and dorsal horn of the spinal cord. KL protein was identified in cells complementary to staining for the receptor, such as glandular myoepithelium of breast and sweat glands. Intense KL immunoreactivity was observed in smooth muscle cells of the bladder, cervix, uterus, and gastrointestinal tract, as well as in striated and cardiac muscle. Strong KL staining was also detected in prostate fibromuscular stroma cells. In the central nervous system, KL expression was confined to Golgi and Purkinje cells in the cerebellum. These results suggest a role for this receptor and its ligand in the maintenance of a variety of fully differentiated tissues.
Distribution of hyaluronan in bull reproductive organs.
Authors: Tammi R , Rönkkö S , Agren U M , Tammi M
Published: April 2011

To study the expression of hyaluronan in male reproductive organs and the origin of seminal plasma hyaluronan, we stained various parts of the bull reproductive tract for hyaluronan using a biotinylated probe derived from cartilage proteoglycan (bHABC). The potential loss of hyaluronan during tissue processing was checked with a novel technique by blotting frozen tissue sections on nitrocellulose and staining the blots with bHABC. In the same tissues the CD44 receptor was visualized by Hermes 1 antibody. The testes showed only traces of hyaluronan, whereas both the epithelium and the connective tissue of seminal vesicle, prostate, Cowper's gland, and epididymis were positive in bHABC staining. Hyaluronan was localized on the basolateral surfaces of these epithelial cells. The secretions inside the seminal vesicle and in the ducts of prostate and Cowper's gland were HA-positive, whereas the luminal contents of seminiferous tubules and epididymis were unstained both in paraffin sections and in the in situ blocks. The data indicate that hyaluronan in seminal plasma originates from the accessory sex glands. The co-localization of CD44 with hyaluronan in the basolateral surfaces of the accessory gland epithelia and its absence from other epithelia with little or no hyaluronan supports its role as a hyaluronan receptor.
Immunohistochemical localization of procollagen types I and III during placentation in pregnant rats by type-specific procollagen antibodies.
Authors: Kitaoka M , Iyama K , Yoshioka H , Monda M , Usuku G
Published: April 2011

To examine the sequential localizations of procollagen Types I (Pro I) and III (Pro III) during chorioallantoic placental formation in pregnant rats, we prepared polyclonal anti-rat Pro I- and III-specific antibodies. Biochemical analysis of a fraction containing [14C]-glycine-incorporated collagen from pregnant rat uteri showed that collagen Types I and III were actively synthesized during placental development. We examined 8-, 9.5-, 13-, and 20-day gestation rat uteri immunohistochemically. At Days 8 and 9.5, in the basal decidua facing the fetal cytotrophoblastic giant cell layer and implantation site, the immunoreactivity for Pro I was higher than that for Pro III. On Day 13, the enlarged myometrium and cytotrophoblastic cell layer showed increased immunoreactivity for Pro III. Unexpectedly, polygonal trophoblastic cells invading and modifying the maternal central artery showed intense immunoreactivity for Pro III. On Day 20, the fetal mesenchyme, large fetal blood vessels, and subendothelial stroma, including fetal blood capillaries, were more immunoreactive to Pro III antibody than to Pro I antibody in the labyrinth. Pro I and III synthesis and processing appear to be developmentally regulated and may be related to control of the microenvironment for supporting the fetus, control of the maternal blood supply stabilizing the fetoplacental physiological functions, and parturition.
Visualization by an atomic force microscope of the surface of ultra-thin sections of rat kidney and liver cells embedded in LR white.
Authors: Yamamoto A , Tashiro Y
Published: April 2011

We visualized the surface of ultra-thin sections of rat liver and kidney embedded in LR White resin using an atomic force microscope. The section surface always showed depressions corresponding to the embedded cells at the plasma membrane, at the basal lamina, at mitochondria, and chromatin blocks in the nucleus. The depth of the depression corresponding to the plasma membrane was about 6 nm in both hepatocytes and several types of kidney cells. At the basal lamina, mitochondria, and the chromatin blocks, sections showed deeper depressions of about 10-30 nm. In addition, cytoplasmic surfaces showed strong relief, 3-4 nm on average. The surfaces of the resin block left after ultrathin sectioning showed protrusions corresponding to cells, mitochondria, and the chromatin blocks. In marked contrast, the surface of epon sections was much smoother than that of LR White sections, without showing any marked depressions and relief except for the chromatin blocks of the nucleus. The relation of the surface morphology of the ultrathin sections to the efficiency of immunolabeling is discussed.
Localization of cystatin mRNA in chicken brain by in situ hybridization.
Authors: Colella R , Kaplan I , Mower G D
Published: April 2011

The cystatins are a superfamily of proteins that inhibit the lysosomal cysteine proteinases, cathepsins B, H, and L. Members of this superfamily have been found in all tissues and biological fluids analyzed. Previous studies have shown that chicken cystatin mRNA is abundant in brain tissue. In this study, a definitive localization of chicken cystatin mRNA in chicken brain was determined by in situ hybridization. Chicken cystatin mRNA was heavily concentrated in the secretory epithelial cells of the choroid plexus. The rest of the brain failed to show a hybridization signal above that of the control sense strand probe, even after long exposures. We conclude that chicken cystatin is synthesized predominantly by the specialized secretory epithelial cells of the choroid plexus and secreted into the cerebrospinal fluid (CSF). We postulate that chicken cystatin functions to regulate proteinase activity in the CSF and therefore may function as a protective factor for the cellular elements of the central nervous system.
Introduction of a fast and sensitive fluorescent in situ hybridization method for single-copy detection of human papillomavirus (HPV) genome.
Authors: Siadat-Pajouh M , Ayscue A H , Periasamy A , Herman B
Published: April 2011

At present, in situ hybridization (ISH) is the only method for detection of specific genes in morphologically intact cells or tissue. We have developed a highly sensitive and quantitative fluorescence-based in situ hybridization (FISH) technique that can detect as few as one to five copies of the integrated human papillomavirus (HPV) type 16 genome in cervical cell lines, using digoxygenin tail-labeled oligonucleotides (Method 1). The entire procedure can be carried out in 4.5 hr through the elimination of some of the steps routinely used in other ISH protocols. We also compared the sensitivity of this new FISH method (Method 1) to four other FISH techniques: digoxigenin-labeled DNA probe (Method 2); fluorescein-15-d-ATP-labeled oligonucleotides (Method 3); fluorescein 15-d-ATP labeled DNA probe (Method 4); and biotin-DNA-labeled probe (Method 5), for their ability to detect HPV DNA in the HPV-positive human cervical cell lines CaSki (500 copies) and SiHa (1-5 copies), but not in C33-A and HT-3, which do not contain any copies of HPV. Our results indicate that Method 1 is more sensitive than the other methods employed. Method 1 was the only method that could reliably detect an HPV-16 genome in all SiHa cells. Our data suggest that the Method 1 FISH technique is highly sensitive and may therefore be of general use for detection and quantitation of a variety of viral genomes (including HIV), oncogenes, and drug-resistant genes, in a variety of morphologically intact cells and tissues.
Quantification of Ag-NOR proteins using Ag-NOR staining on western blots.
Authors: Roussel P , Sirri V , Hernandez-Verdun D
Published: April 2011

Ribosomal genes are associated with a set of silver-stained nucleolar proteins, the Ag-NOR proteins, whose amount is directly related to the duration of the cell cycle. Quantification of Ag-NOR proteins by image analysis is presently used to evaluate the rate of proliferation of cancer cells and nucleolar activity. Our objective was to establish a procedure to quantify independently each major Ag-NOR protein in cell extracts. Computerized densitometry established that the specific silver staining of Ag-NOR proteins (Ag-NOR staining) performed on Western blots makes it possible to quantify Ag-NOR proteins. Using purified Ag-NOR proteins, nucleolin, and protein B23, we observed that the intensity of Ag-NOR staining is proportional to the amount of protein. A linear relationship exists between the intensity of Ag-NOR staining and the amount of nucleolin, in the range of 0.2-1.6 micrograms. Using total nuclear extracts prepared from mammalian cells, the proportionality was maintained for total Ag-NOR-stained proteins or for a particular protein. We also determined the levels of nuclear proteins suitable for quantitative analysis. Individual Ag-NOR proteins can be quantified by computerized densitometry in nuclear extracts after Ag-NOR staining on Western blots. This procedure can be applied to establish the contribution of each Ag-NOR protein in general staining, estimate the variability of each Ag-NOR protein in normal and pathological conditions, and quantify each Ag-NOR protein contained per cell.
Roles of Triton X-100 in NADPH-diaphorase histochemistry.
Authors: Fang S , Christensen J , Conklin J L , Murray J A , Clark G
Published: April 2011

Triton X-100 is widely but not universally used in NADPH-diaphorase histochemical staining. We investigated its effect on the staining and examined nitroblue diformazan (NBF) production under the influence of Triton X-100. Exposure of opossum esophagus, intestine, and colon tissues to Triton X-100 before staining enhanced staining of nerve cells and fibers and suppressed staining of non-neural structures. Long exposures and high concentrations nearly abolished the staining of non-neural structures and decreased the staining of nerves. The use of an incubation medium containing Triton X-100 achieved the best staining of nerve cells and fibers. Addition of Triton X-100 to the incubation medium changed its color from yellow to purple; in the presence of tissues, this color change occurred much more quickly. Spectral analysis showed that Triton X-100 increases the rate of NBF formation in the presence of tissue supernatant. Triton X-100 increases it less in the absence of tissue supernatant. Therefore, Triton X-100 improves the histochemical staining, probably by catalyzing the activity of NADPH-diaphorase, by keeping the extracellular NBF in solution and thus suppressing the staining of non-neural structures, and by increasing the permeability of cell membranes.
Novel in situ double labeling for simultaneous detection of proliferation and apoptosis.
Authors: Mundle S , Iftikhar A , Shetty V , Dameron S , Wright-Quinones V , Marcus B , Loew J , Gregory S , Raza A
Published: April 2011

We describe a novel double-labeling method to simultaneously investigate proliferation and apoptosis from plastic-embedded biopsy specimens (PEBs). Infusions of bromo- and/or iododeoxyuridine (BrdU/IudR) were given to 10 patients, five with acute myeloid leukemia (AML) and five with myelodysplastic syndromes (MDS), and S-phase cells were measured in PEBs using a monoclonal anti-IudR/BrdU antibody. Apoptosis was measured by in situ end-labeling (ISEL) of DNA. The results demonstrate that both AML and MDS are highly proliferative disorders but that there is almost no apoptosis in the former, whereas extensive apoptosis was observed in the latter. Double labeling revealed that large numbers of S-phase cells in MDS were simultaneously undergoing apoptosis. We conclude that the high cell death in MDS cancels the high cell birth, resulting in a functionally aplastic marrow and thus accounting for the observed ineffective hematopoiesis. On the other hand, AML is rapidly fatal, probably owing to high cell birth with no or minimal cell death. Therapeutic strategies to prevent intramedullary programmed cell death of hematopoietic precursors should be evaluated in MDS, and efficacy of chemotherapy in AML can be assessed by measuring the induction of apoptosis in post-treatment biopsy specimens.
Filipin vs enzymatic localization of cholesterol in guinea pig, mink, and mallard duck testicular cells.
Authors: Pelletier R M , Vitale M L
Published: April 2011

To test the validity of filipin cytochemistry for localization of cholesterol in testicular cells, we compared the results obtained by this technique with those obtained by a two-step enzymatic method involving cholesterol esterase and cholesterol oxidase. In all the animals models tested (guinea pig, mink, and mallard duck) the disappearance of subsurface filaments along Sertoli cell junctional membranes was accompanied by a significant increase in the number of filipin-cholesterol complexes/microns 2 in these membranes. Enzyme histochemistry allowed localization of free cholesterol in the limiting membrane of multivesicular bodies, in membranes within lysosomes, in mitochondrial membranes, and in junctional membranes, with or without subsurface filaments. The method also permitted selective visualization of cholesterol esters in lipid droplets. We conclude that filipin mapping of cholesterol induces false-negative cytochemical results. The enzymatic method is superior to filipin because it allows localization of free cholesterol in junctional membranes and of cholesterol esters in lipid droplets. This compartmentalization of the compounds may represent the basis of a system that helps to maintain constant free cholesterol levels in the testis.
cFos immunoreactivity is enhanced with biotin amplification.
Authors: Berghorn K A , Bonnett J H , Hoffman G E
Published: April 2011

Through modification of the protocol by Adams, we developed a biotin amplification procedure for immunofluorescence staining of immediate early gene proteins and also applied biotin amplification for metal enhancement of diaminobenzidine staining in an immunoperoxidase protocol. Commercially available anti-cFos antisera were used to compare conventional "Elite" avidin-biotin complex reactions with biotin amplification reactions (visualized with peroxidase staining or streptavidin-Texas Red fluorescence). Biotin amplification and peroxidase staining (with or without nickel salts) enabled detection of cFos in stimulated neurons with primary antibody concentrations five- to tenfold lower than the conventional procedure. With immunofluorescence staining, at primary antibody concentrations too low to detect cFos with the conventional biotin-streptavidin fluorescence staining protocol, biotin amplification enabled clear cFos fluorescence staining with both antisera. The fluorescence staining exhibited a high signal-to-noise ratio and enabled antibody concentrations four times lower than those used for conventional ABC "Elite" peroxidase procedures. In conclusion, the application of biotin amplification to cFos immunocytochemical localization has the promise of aiding the scientist in detecting these immediate early gene products.
An alternative method for preparation of Schiff-like reagent from osmium-ammine complex for selective staining of DNA on thin Lowicryl sections.
Authors: Mikhaylova V T , Markov D V
Published: April 2011

Batches of osmium-ammine (OA) complex vary considerably in staining properties when used in a Feulgen-like reaction for selective staining of DNA-containing structures. An alternative procedure for preparation of Schiff-like reagent from OA complex is described. It is based on generation of H2SO3, respectively SO2, within the OA solution and ensures more favorable conditions for production of a Schiff-like stain than bubbling with SO2 does. The method is reproducible and yields high staining intensity. Data obtained suggest that the ability of OA complex to produce Feulgen-like staining is strongly influenced by variations in its chemical composition. Their unfavorable effect can be overcome by selecting suitable conditions for preparation of a Schiff-like reagent. Conditions for obtaining specific and sensitive Feulgen-like staining are determined.
Demonstration of acidity in intestinal vacuoles of the suckling rat and pig.
Authors: Baintner K
Published: April 2011

Fluorescence staining characteristics of "large vacuoles," i.e. vacuoles ranging up to almost cell size, were studied in suckling rats and pigs. In the distal epithelium of the small intestine of suckling rat, yellow autofluorescence and accumulation of orally administered FITC-dextran were observed in the supranuclear vacuole. In both species the weakly basic amino dye acridine orange (AO) stained the nuclei at neutral pH bright yellow-green and the transport and digestive vacuoles bright red or orange. It is concluded that trapping and accumulation of the dye (red shift) were due to the acidity of the vacuolar interior. Assessment of the vacuolar pH in rat enterocytes is in agreement with published data on lysosomal pH values. Acidic buffers, lysosomotropic and destructive agents, or illumination with bright light induced irreversible fading of AO-stained vacuoles; the color of the porcine transport vacuoles was the most labile. This fading was used to differentiate vacuoles from other structures, e.g., vacuolar inclusion bodies and goblet cells. In suckling rat, staining characteristics of the gut epithelium changed on Days 19 and 20 of postnatal age. Detection of acidity in the distal (digestive) vacuoles supports the lysosome-like nature of their function. They appear to constitute an auxiliary, intracellular digestive system for the young animal. However, the function of acidity in the non-digestive transport vacuoles of newborn pig is unclear.
Distribution of beta-1 integrin in the developing rat heart.
Authors: Carver W , Price R L , Raso D S , Terracio L , Borg T K
Published: April 2011

Cell-cell and cell-matrix interactions play critical roles in various developmental processes including differentiation, proliferation, and migration. Members of the integrin family of cell surface components are important mediators of these cell-extracellular matrix (ECM) contacts or interactions. The ECM provides signals to individual cells essential for development and differentiation and plays essential roles in establishing and maintaining the complex structure of the vertebrate heart. Integrins provide a fundamental link for transduction of developmental signals to cells. Integrin expression by cardiac myocytes is altered during neonatal heart development and disease; however, little is known regarding the spatial and temporal patterns of integrin expression during embryonic and fetal heart development. Essential to understanding the role of integrins in the organization of the heart, the present studies have localized beta-1 integrin protein and mRNA in fetal and neonatal rat hearts. Beta-1 integrin is predominantly found in regions of remodeling (trabeculae) in the early heart (10-13 days of gestation). Later in development (15 days of gestation onward), beta-1 integrin is abundant in regions containing an elaborate ECM, such as the valves. These studies further support the hypothesis that the expressions of integrins and ECM are coordinately regulated in the developing heart.
Localization of uric acid oxidase activity in core and matrix of peroxisomes as detected in unfixed cryostat sections of rat liver.
Authors: Van den Munckhof R J , Vreeling-Sindelárová H , Schellens J P , Frederiks W M
Published: April 2011

Because of controversial data in the literature, we studied the localization of uric acid oxidase (UAOX) activity in rat liver by light microscopy (LM) and electron microscopy (EM). UAOX is partially inactivated by aldehyde fixation and therefore we developed a technique that permits the use of unfixed cryostat sections for both LM and EM studies. Sections of rat liver were mounted on a semipermeable membrane stretched over a gelled incubation medium containing urate as specific substrate for UAOX and cerium ions to capture H2O2 produced by oxidase activity. The specificity of the reaction was checked by comparing incubations in the presence of substrate with incubations either in the absence of substrate or in the presence of substrate and 2,6,8-trichloropurine, a competitive inhibitor of UAOX. After incubation the sections were either fixed immediately for EM or visualized for LM with a second-step incubation. At the LM level, final reaction product was found in a granular form, homogeneously distributed throughout the hepatocytes. EM revealed excellent subcellular morphology and electron-dense reaction product in both the core and the matrix of peroxisomes, but not in other organelles or the cytoplasmic matrix. After incubations without substrate or with substrate and inhibitor, hardly any reaction product was found. We conclude that, because of the use of unfixed tissue, UAOX is not inactivated, which results in localization of UAOX activity not only in the core of peroxisomes but also in the peroxisomal matrix.
Cell phenotypes and differentiative transitions in mouse submandibular salivary gland defined with monoclonal antibodies to mammary epithelial cells.
Authors: Durban E M , Barreto P D , Hilgers J , Sonnenberg A
Published: April 2011

Recognition of distinct cell phenotypes within a given organ is important in defining cell relationships during development and in analyzing the role of cell-cell and cell-matrix interactions in growth and differentiation. Phenotypic definition of dissociated heterogeneous cell populations is also essential for studies on mechanisms regulating expression of cell lineage-specific gene products. Mouse submandibular salivary gland (SSG) cell phenotypes in the course of differentiative transitions in vivo and after enzymatic dissociation in primary culture were defined with monoclonal antibodies (MAb) to mammary epithelial cells and polyclonal antibodies to functional cell products. Proacinar cells and differentiating and mature acinar cells were uniquely recognized by an MAb designated 50B8. Ductal cell components were uniquely recognized by an MAb designated JSE3. JSE3 immunoreactivity was particularly useful for detecting the emergence of two SSG duct cell phenotypes, striated ducts and the hormone-responsive granular convoluted tubules (GCTs). JSE3-positive striated duct-like cells were visualized as early as Day 2 after birth and emergence of GCT-like structures from striated ducts was apparent between Days 10 and 11. Differential reactivity of acinar and ductal cells in the developing SSG with either MAb 50B8 or JSE3 suggests the existence of intermediate progenitor cells restricted in their differentiation potential. An interesting pattern of immunoreactivity was observed with an MAb designated 33A10. During the first 2 weeks of SSG postnatal development, shared 33A10 immunoreactivity was observed among proacinar and differentiating acinar cells and all differentiating ductal segments. Coincident with a decrease in proliferative activity at about Day 18, 33A10 immunoreactivity became restricted to the GCT cell lineage before the appearance of GCT functional products, epidermal and nerve growth factors. Although the SSG antigen recognized by MAb 33A10 is presently undefined, its expression pattern suggest a molecule with a dual role in development and growth events and in hormone-dependent secretory function. Advantage was taken of the observed differential immunoreactivities to define the phenotypic identity of dissociated, mature SSG cells before and after culture. Dissociated SSG fractions enriched for either JSE3- or 50B8-positive cells could be maintained in short-term cultures without loss of expression of duct- or acinar cell-specific immunoreactivity. In addition to providing markers for defining dissociated SSG cells before and after culture, the described immunoreactivities may permit separation or enrichment of an early duct cell population before its commitment to a specific cell lineage. This approach may also provide the means to define regulatory signals involved in the differentiation of intermediate progenitor cells.