bluuebunny/crossref_metadata_2025 huggingface dataset:
Retrograde transport of the lectin Phaseolus vulgaris leucoagglutinin in frog central nervous system.
Authors: Antal M , Petkó M
Published: April 2011

The transport properties of the lectin Phaseolus vulgaris leucoagglutinin (PHA-L) were tested in the frog central nervous system. After delivery of the lectin to the lower brainstem by iontophoresis, stained axons and axon terminals, as well as neurons with richly arborizing dendrites, were observed indifferent regions of the brain and spinal cord even far away from the site of application. The large number and the Golgi-like appearance of labeled neurons situated rostral and caudal to the site of PHA-L deposit indicate that PHA-L is transported equally in both the anterograde and the retrograde direction in the central nervous system of the frog. This is in contrast with the mammalian nervous system, in which PHA-L is transported predominantly in the anterograde direction and the retrograde transport is poor.
Immunocytochemistry of glutamate at the synaptic level.
Authors: Storm-Mathisen J , Ottersen O P
Published: April 2011

High concentrations of glutaraldehyde (2-5%) were found optimal for fixation of glutamate. In the absence of glutaraldehyde, (para)formaldehyde does not permanently retain L-[3H]-glutamate or D-[3H]-aspartate previously taken up into brain slices. Rats were fixed by rapid transcardial perfusion with 2.5% glutaraldehyde/1% (para)formaldehyde, and brain samples osmicated, embedded in epoxy resin, sectioned, and exposed to specific antisera to glutamate (conjugated to carrier protein by glutaraldehyde), followed by colloidal gold-labeled second antibody. The gold particle density was higher over putative glutamatergic nerve terminals than over any other tissue elements (two to three times tissue average in cerebellum and hippocampus). Calibration by test conjugates containing known concentrations of fixed glutamate processed in the same fluid drops as the tissue sections indicated that the concentration of fixed glutamate in putative glutamatergic terminals in hippocampus CA1 was c. 20 mmol/liter. The grain density over the parent cell bodies was only slightly higher than the tissue average. (Grain densities over test conjugates of other amino acids, aldehyde-fixed to brain macromolecules, were similar to that over empty resin. Labeling was blocked by glutamate-glutaraldehyde but not by other glutaraldehyde-treated amino acids.) In other experiments, brain slices were incubated in oxygenated artificial cerebrospinal fluid (CSF) and then immersion-fixed and processed as above. Here, the ration of grain densities in putative glutamatergic terminals vs other tissue elements was greater than in perfusion-fixed material. Comparison of intra-terminal areas poor and rich in synaptic vesicles suggested that in this preparation vesicles contained at least three times the glutamate concentration of cytosol. In the glutamatergic synapses of the giant reticulospinal axons in lamprey the ratio was over 30. Prolonged K+ depolarization of hippocampal and cerebellar slices reduced the nerve terminal glutamate immunoreactivity in a Ca2(+)-dependent manner. The results suggest that glutamate is released by exocytosis at excitatory synapses and show that immunocytochemistry can be used to study the cellular processing of small molecules.
Isolation, localization, and cloning of a kainic acid binding protein from frog brain.
Authors: Wenthold R J , Hampson D R , Wada K , Hunter C , Oberdorfer M D , Dechesne C J
Published: April 2011

Excitatory amino acids (EAA) are major neurotransmitters in the vertebrate central nervous system. EAA receptors have been divided into three major subtypes on the basis of electrophysiological and ligand binding studies: N-methyl-D-aspartate, kainate, and quisqualate receptors. To understand their molecular properties, we undertook a project aimed at isolation and cloning of these receptor subtypes. We purified a kainate binding protein (KBP) from frog brain, in which kainate binding sites are about fortyfold more abundant than in rat brain, using domoic acid affinity chromatography, and made monoclonal and polyclonal antibodies to the purified protein. These antibodies immunoprecipitate the frog KBP but not KBPs from other species. Immunocytochemical analyses show that KBP has a synaptic and extrasynaptic localization in frog optic tectum, with most labeling being extrasynaptic. The cDNA encoding frog brain KBP was isolated by screening a frog brain cDNA library with oligonucleotide probes that were based on the amino acid sequence of the purified protein. The deduced amino acid sequence of the KBP has a hydrophobic profile similar to those of other ligand-gated ion channel subunits, such as the nicotinic acetylcholine receptor, the GABAA receptor, and the glycine receptor. Frog brain KBP is very similar (36% amino acid identity to the carboxyl half) to rat brain kainate receptor, suggesting that these two proteins evolved from a common ancestor. The function of KBP in frog brain remains a major question. Preliminary results showed that Xenopus laevis oocytes injected with KBP RNA did not produce a detectable electrophysiological response when perfused with kainate. These results suggest that additional subunits may be required to form a functional receptor or that KBP is not functionally related to a neurotransmitter receptor.
Detection of ouabain-insensitive H(+)-transporting, K(+)-stimulated p-nitrophenylphosphatase activity in rat gastric glands by cerium-based cytochemistry.
Authors: Kobayashi T , Seguchi H
Published: April 2011

We employed a modification of our previously reported cerium-based cytochemical method for ouabain-sensitive, K-dependent p-nitrophenylphosphatase (Na-K ATPase) activity to detect ouabain-insensitive, K-stimulated p-nitrophenylphosphatase (K-pNPPase) activity in rat gastric glands. Biochemically, the enzyme activity of gastric glands incubated in a medium containing 50 mM Tricine buffer (pH 7.5), 50 mM KCl, 10 mM MgCl2, 2 mM CeCl3, 2 mM p-nitrophenylphosphate (pNPP), 2.5 mM levamisole, 10 mM ouabain, and 0.00015% Triton X-100, was optimal at pH 7.5-8.0 and decreased above pH 8.5. The amount of p-nitrophenol after incubation increased linearly in proportion to the amount of tissue in the medium. The enzyme activity was inhibited by omeprazole, sodium flouride (NaF), N-ethylmaleimide (NEM), and dicyclohexylcarbodiimide (DCCD). Heat-treated specimens had no enzyme activity. The enzyme activity increased with addition of K ions up to the concentration of 50 mM, and became constant above 50 mM. Cytochemically, the parietal cells of the gastric glands reacted positively for ouabain-insensitive K-pNPPase activity. Intense reaction was observed at the microvilli of the luminal surface and the intracellular canaliculi. The tubulovesicular system showed weak enzyme activity. The reaction products were found as fine, granular, electron-dense deposits in the cytoplasm just beneath the plasma membrane. The ouabain-insensitive K-pNPPase activity detected in this study appears, therefore, to be associated with that of H-transporting, K-stimulated adenosine triphosphatase (H-K ATPase).
Light microscopic autoradiography for study of early changes in the distribution of water-soluble materials.
Authors: Kawamoto T
Published: April 2011

An approach using autoradiography for the study of early changes in the distribution of water-soluble materials and the chemography involved was investigated. Radioactive calcium chloride (45Ca) was injected into the iliac vein of a rat. Ten seconds after the injection the rat was frozen in hexane (-90 degrees C). The frozen rat was embedded in 5% sodium carboxymethyl cellulose and blocked in the coolant. A sheet of plastic tape coated with a synthetic rubber glue was fastened to the trimmed block surface, and whole-body sections 2-10 microns thick were cut with a disposable microtome knife. Selected sections were freeze-dried and then covered with a dried autoradiographic emulsion film about 1 microns thick. The autoradiograph clearly showed the distribution of radioactive calcium in the calcification zone of long bones. The samples chosen to assess chemographic artifacts showed positive and negative chemographies on most of the tissues when these were kept at 23 degrees C, and although both chemographic effects were significantly reduced when the samples were kept at -20 degrees C, cells in several tissues still exhibited positive and negative chemographies. The technique can be used for the study of any animal whose size is suitable for whole-body freeze-sectioning.
A novel polyclonal antibody (CL-B1/29) for immunolocalization of transforming growth factor-beta 2 (TGF-beta 2) in adult mouse.
Authors: Ksander G A , Gerhardt C O , Dasch J R , Ellingsworth L R
Published: April 2011

A polyclonal antibody (CL-B1/29) raised against a synthetic peptide with an amino acid sequence identical to the first 29 N-terminal residues of bovine bone-derived transforming growth factor-beta 2 (TGF-beta 2) was characterized and used for immunolocalization of TGF-beta 2 in adult mice. Reduced staining of immunoblots and tissue after absorption of the antiserum with the immunizing peptide or with TGF-beta 2 but not with purified TGF-beta 1 demonstrated that the reagent is specific for TGF-beta 2, with little or no crossreactivity with TGF-beta 1. The immunolocalization of TGF-beta 2 was investigated in formalin-fixed, paraffin-embedded cultured cells and murine tissue. Specimens pre-digested with testicular hyaluronidase demonstrated immunostaining predominantly of extracellular connective tissue matrix, whereas specimens pre-digested with pronase E demonstrated primarily cytoplasmic staining. Immunoreactivity was widely distributed in connective tissue, muscle, adsorptive and secretory epithelia, especially of endocrine tissue, and neural tissue of adult mice.
Immunohistochemical detection of cytochrome P450 isoenzymes in cultured human epidermal cells.
Authors: Van Pelt F N , Meierink Y J , Blaauboer B J , Weterings P J
Published: April 2011

We used specific monoclonal antibodies (MAb) to human cytochrome P450 isoenzymes to determine the presence of these proteins in human epidermal cells. Two MAb (P450-5 and P450-8) recognize major forms of hepatic cytochrome P450 involved in biotransformation of xenobiotics. A third MAb, to cytochrome P450-9, is not fully characterized. The proteins were determined by the indirect immunoperoxidase technique after fixation with methanol and acetone. Biopsy materials for cultured keratinocytes, i.e., foreskin and hair follicles, contained the two major forms of cytochrome P450. In cultured keratinocytes derived from hair follicles the proteins were undetectable, whereas the keratinocytes derived from foreskin continued to express the two major forms of hepatic cytochrome P450. Cultured human fibroblasts and a human keratinocyte cell line (SVK14) showed staining similar to that of the foreskin keratinocytes. Cytochrome P450-9 was detectable only in human hepatocytes. The results indicate that, under the culture conditions applied, cultured human foreskin cells and the cell line SVK14 continue to express specific cytochrome P450 isoenzymes in culture, in contrast to hair follicle keratinocytes.
An enhanced method for post-embedding immunocytochemical staining which preserves cell membranes.
Authors: Berryman M A , Rodewald R D
Published: April 2011

We have devised a method for immunogold staining of unosmicated, plastic-embedded tissue which gives high levels of specific staining without scrificing cell ultrastructure. The key to this method is a combination of several standard techniques optimized to preserve cell membranes as well as antigen. Important conditions include (a) a combination primary fixative, (b) post-fixation with uranyl acetate to preserve membrane phospholipids, (c) dehydration with acetone to minimize extraction of phospholipids, (d) low-temperature embedding in LR Gold resin, and (e) use of osmium tetroxide to stain thin sections after immunogold labeling. We have developed this method specifically to localize the membrane receptor for immunoglobulin G in the jejunal epithelium of the neonatal rat. Ultra-thin sections of embedded tissue were stained with a monoclonal primary antibody and colloidal gold-labeled secondary antibody, followed by 2% osmium tetroxide and lead citrate. The receptor was resolved in the well-preserved network of tubules, endosomes, and other membrane compartments involved in immunoglobulin transport. In several other tissues processed by this method, cell ultrastructure resembled that seen after conventional osmium post-fixation and epoxy embedding. In addition to its usefulness in these studies, this general method should be applicable to many other immunocytochemical problems.
Immunohistochemical localization of polysialic acid in tissue sections: differential binding to polynucleotides and DNA of a murine IgG and a human IgM monoclonal antibody.
Authors: Husmann M , Roth J , Kabat E A , Weisgerber C , Frosch M , Bitter-Suermann D
Published: April 2011

For immunolocalization of alpha(2-8)-linked polysialic acid, which forms part of the neural cell adhesion molecule (N-CAM), two monoclonal antibodies, MAb735 and IgMNOV, were employed. Both antibodies have previously been shown to bind the extremely low immunogenic capsular polysaccharide of group B meningococci, which also consists of alpha(2-8) polysialic acid, but not to other, even closely related forms of polysialic acid. Despite the identical polysaccharide specificity of these two MAb, we observed marked differences of the staining pattern in tissue sections. We showed that these differences in immunostaining were due to the crossreactivity of IgMNOV with polynucleotides and DNA. MAb735, however, was shown to react exclusively with alpha(2-8) polysialic acid. Moreover, the specificity of MAb735 proved to be unique among eleven other MAb directed against various bacterial polysaccharides, as it was the only one unreactive with polynucleotides. Thus, MAb735, the only IgG type mouse monoclonal antibody to polysialic acid thus far reported, can be considered a specific probe for the unambiguous detection of alpha(2-8) polysialic acid in tissue sections, and should therefore help to further elucidate the role of polysialic acid in developmental processes.
The presence of a type IV collagen skeleton associated with periductal elastosis in breast cancer.
Authors: Verhoeven D , Bourgeois N , Noël A , Foidart J M , Buyssens N
Published: April 2011

Using serial sections of frozen and AFA-fixed tissues from 34 breast cancers, we studied the presence of basement membrane material in the areas of elastosis. Various amounts of type IV collagen but not of laminin were demonstrated in areas of periductal elastosis. In some tumors, type IV collagen accumulated beneath the basement membrane. Periductal elastosis in areas of extensive fibrosis showed focal type IV collagen immunoreactivity, indicating remnants of ducts. Interstitial elastosis corresponded with weak type IV collagen reactivity. Each tumor showed type IV collagen immunostaining of the elastotic areas, with various degrees of intensity. Negative crossreactivity of the type IV collagen antibody with elastin was verified in skin biopsies with solar elastosis. Pre-incubation of the antibody with large amounts of elastin demonstrated an identical immunoreactivity. The specificity of the antibody was confirmed by ELISA and by Western blot analysis. To explain the periductal elastosis, we propose the following hypothesis. Excessive production of basement membrane material by the epithelial cells of the ducts leads to formation of a type IV collagen skeleton. This skeleton can act as the matrix for a secondary deposition of elastic material.
Identification of a novel type 2 fiber population in mammalian skeletal muscle by combined use of histochemical myosin ATPase and anti-myosin monoclonal antibodies.
Authors: Gorza L
Published: April 2011

A novel type of myosin heavy chain (MHC), called 2X, has been recently identified in type 2 fibers of rat skeletal muscles using an immunochemical approach. In the present study, the same panel of anti-MHC monoclonal antibodies was used in immunohistochemistry combined with enzyme histochemistry to identify and compare type 2X fibers in hindlimb skeletal muscles of rat, mouse, and guinea pig. Immunohistochemistry shows that 2X MHC is localized in a large subset of type 2 fibers and is co-expressed with 2A or 2B MHC in a small number of fibers. Enzyme histochemistry shows that type 2X fibers display low myosin ATPase activity after pre-incubation at pH 4.3 and high activity after paraformaldehyde pre-incubation at pH 10.4. After pre-incubation at pH 4.6, myosin ATPase shows intermediate and high activity in rat and mouse 2X fibers, respectively, whereas it is low in guinea pig 2X fibers. Succinate dehydrogenase displays moderate to high activity in 2X fibers of all species. Taken together, these staining patterns allow this novel fiber population to be distinguished from the other type 2 fibers using only enzyme histochemistry. Nevertheless, the combined use of immuno- and enzyme histochemistry prevents incorrect fiber typing due to the interspecies variability of myosin ATPase activity among the correspondent fiber types, and completely modifies the presently used classification of mouse type 2 fibers.
Quantitative immunogold localization of Na+,K(+)-ATPase alpha-subunit in the tympanic wall of rat cochlear duct.
Authors: Iwano T , Yamamoto A , Omori K , Kawasaki K , Kumazawa T , Tashiro Y
Published: April 2011

Ultrastructural localization of Na+,K(+)-ATPase was quantitatively investigated in the tympanic wall of rat cochlear duct by use of the protein A-gold method, using an affinity-purified antibody against the alpha-subunit of rat kidney Na+,K(+)-ATPase. A moderate number of gold particles were found on the basolateral membrane of the interdental cells of the spiral limbus. A small number of gold particles were found on the basolateral surfaces of the border cells and Hensen's cells. On the inner and outer sensory hair cells, however, the plasma membranes were rarely labeled by gold particles. The general pattern of labeling densities in cochlear structures determined here and in a previous communication from our laboratory shows good correlation with the distribution of Na+,K(+)-ATPase activity as previously estimated biochemically, cytochemically, and autoradiographically.
Ultrastructural distribution of terbium across capillary endothelium: detection by electron spectroscopic imaging and electron energy loss spectroscopy.
Authors: Wagner R C , Chen S C
Published: April 2011

We used terbium as an intravital tracer of permeability pathways across the walls of capillaries in the rete mirabile of the eel swimbladder and in frog mesentery. Terbium was detected in unstained ultra-thin sections by electron density using electron spectroscopic imaging (ESI) and by electron energy loss spectroscopy (EELS). Enhancement of intrinsic contrast in zero loss images (elastically scattered electrons) permitted imaging of membrane-bound compartments and terbium within them which might otherwise have been undetected in counterstained sections. Element-selective imaging with EELS indicated that terbium was associated with heavy electron-dense deposits, but the terbium mass:volume of sections in areas of lighter deposition was insufficient to obtain a terbium signal. In the rete capillaries, terbium was deposited on the luminal surface, throughout vesicular profiles, and in the interstitium, but could not be traced through interendothelial junctions. Fine terbium deposits were detectable throughout apparent vesicular connections across the endothelium. In the frog mesentery, terbium penetrated some but not all interendothelial clefts, and was detectable in small quantities within luminal and abluminal vesicular profiles and in the interstitium. The results indicate that in the rete capillaries, terbium permeates the capillary via a transcellular route. This route may be provided by transient fusions of luminal and abluminal vesicular compartments.
Albumin and collagen mRNA expression in normal and analbuminemic rodent liver: analysis by in situ hybridization using biotinylated probes.
Authors: Saber M A , Novikoff P M , Shafritz D A
Published: April 2011

We used in situ nucleic acid hybridization cytochemistry to examine cell types and subcellular sites expressing albumin (alb) or pro alpha 2 collagen (col) mRNA in livers from normal and analbuminemic rodents. Biotinylated cDNA or RNA probes were applied to aldehyde-fixed, non-frozen sections and the resulting DNA-RNA or RNA-RNA hybrids were subsequently visualized by enzymatic detection of either peroxidase or alkaline phosphatase conjugated to anti-biotin IgG or streptavidin. In normal rat liver, alb mRNA was expressed in all hepatocytes and was localized to discrete subcellular structures distributed as aggregates in the cytoplasm and in specific structures encircling the nucleus; these subcellular structures most likely represent the endoplasmic reticulum and nuclear envelope. In mouse liver, pro alpha 2 col mRNA was identified in a subpopulation of sinusoidal lining cells which have the morphological appearance of lipocytes. In liver from analbuminemic rats, a small number of hepatocytes, distributed throughout the hepatic lobule, expressed alb mRNA at high levels; the subcellular distribution of this alb mRNA was essentially identical to that observed in normal rat hepatocytes. Since non-radioactive in situ hybridization detected mRNA within the boundaries of individual cells and showed its precise subcellular location under conditions in which there was excellent preservation of tissue morphology, this procedure should be useful for a wide variety of histopathologic studies.
Immunohistochemical localization of glycosaminoglycans and proteoglycans in predentin and dentin of rat incisors.
Authors: Takagi M , Hishikawa H , Hosokawa Y , Kagami A , Rahemtulla F
Published: April 2011

We examined immunocytochemically the type and distribution of glycosaminoglycans and proteoglycans (PG) in predentin and dentin demineralized with EDTA after aldehyde fixation of rat incisors using (a) four monoclonal antibodies (1-B-5,9-A-2,3-B-3, and 5-D-4) which recognize epitopes in unsulfated chondroitin (C0-S), chondroitin 4-sulfate (C4-S), chondroitin 6-sulfate (C6-S), and keratan sulfate (KS) associated with the PG, and (b) monoclonal (5-D-5) and polyclonal antibodies specific for the core protein of large and small dermatan sulfate (DS) PG. Light microscope immunoperoxidase staining after pre-treatment of tissue sections with chondroitinase ABC localized the majority of stainable PG (C4-S, KS, DSPG, C0-S, and C6-S) in predentin and, to a lesser extent (C4-S and small DSPG), in the dentin matrix. The former site demonstrated relatively homogeneous PG distribution, whereas the latter site revealed that strong staining of C4-S and small DSPG was confined mostly to dentinal tubules surrounding odontoblastic processes, with only weak staining in the rest of the dentin matrix. These results indicate that there is not only a definite difference between PG of predentin and dentin but also a selective decrease in the concentration or alteration of these macromolecules during dentinogenesis and mineralization.
Expression of intermediate-sized filaments in developing and adult human kidney and in renal cell carcinoma.
Authors: Oosterwijk E , Van Muijen G N , Oosterwijk-Wakka J C , Warnaar S O
Published: April 2011

We studied the distribution of intermediate-sized filaments in developing and adult kidneys and renal cell carcinoma (RCC) by indirect immunohistochemistry, using a pan-cytokeratin mouse monoclonal antibody (MAb), chain-specific anti-cytokeratin MAb, and anti-vimentin and anti-desmin MAb, to resolve controversy concerning intermediate-sized filament expression in the kidney. With the pan-cytokeratin MAb, cytokeratin expression was detectable in all stages of nephron development, starting with expression in the renal vesicles, the progenitors of the glomeruli, proximal tubules, Henle's loop, and part of the distal tubules. Using chain-specific anti-cytokeratin MAb, cytokeratin 8 and 18 expression was demonstrated in all developmental structures of the nephron, whereas cytokeratin 19 expression was more complex. None of the nephrogenic blastema cells from which the renal vesicles arise expressed cytokeratins. Transient expression of vimentin and cytokeratin 19 was observed in differentiating collecting ducts and proximal tubule cells at the S-shaped stage of nephron development, respectively. In RCC, cytokeratin expression closely resembled that of the mature proximal tubule, i.e., RCC cells expressed cytokeratins 8 and 18. However, in a subset of RCC additional cytokeratin 19 expression was noted. In addition, all except one RCC showed co-expression of cytokeratins and vimentin.
Routine use of backscattered electron imaging to visualize cytochemical and autoradiographic reactions in semi-thin plastic sections.
Authors: Nanci A , Zalzal S , Smith C E
Published: April 2011

The scanning electron microscope (SEM) was used to examine cytochemical and autoradiographic reactions in 2-microns semi-thin sections of tissues conventionally fixed and embedded in various resins. The sections were examined using both the secondary and backscatter modes of the SEM at magnifications within the range attainable with the light microscope. Both modes allowed the imaging of phosphatase reaction product using cerium and lead capture, lectin-gold, and immunogold labeling, with and without silver enhancement, and autoradiography. Backscattered electron imaging (BEI), however, provided images with more contrast and structural details. This approach allows examination of large sections, with more contrast and resolution than the light microscope, and visualization of reactions not visible with this instrument. The improved imaging and the simple and conventional preparation of specimens indicate that BEI can be used routinely to examine tissue organization, cell structure, and the content of the various cell compartments with a resolution approaching that of transmission electron microscopy.
Influence of fixation procedures on the microanalysis of lead-induced intranuclear inclusions in rat kidney.
Authors: Vandeputte D F , Jacob W A , Van Grieken R E
Published: April 2011

Using Laser Microprobe Mass Analysis (LAMMA), we studied the chemical composition of lead-induced intranuclear inclusions in rat kidney tissue prepared by three different wet chemical fixation procedures for transmission electron microscopy. Fixation with glutaraldehyde-Na2S gave the same results as fixation with glutaraldehyde only: a high lead concentration could be detected. Therefore, for lead strongly bound to proteins, precipitation procedures are not essential. Post-fixation with osmium tetroxide drastically changed the composition of the inclusions: the lead concentration decreased substantially, while sodium, calcium, and barium were introduced. The osmium tetroxide fixative was found to be the source of the contamination. It also contained aluminum, and we suggest that other proteins (e.g., in neurofibrillary tangles) might be able to take up Al out of solution and that care must be exercised in interpreting the microanalytical results of osmium-fixed material. For the microanalysis of the lead inclusions, fixation with glutaraldehyde only provides a good compromise between preservation of the ultrastructure and maintenance of the element distribution.
Levels of carbamoyl phosphate synthetase I in livers of young and old rats assessed by activity and immunoassays and by electron microscopic immunogold procedures.
Authors: Martinez-Ramon A , Knecht E , Rubio V , Grisolia S
Published: April 2011

Carbamoyl phosphate synthetase I, the most abundant protein of rat liver mitochondria, plays a key role in synthesis of urea. Because aging affects some liver functions, and because there is no information on the levels of carbamoyl phosphate synthetase I during aging, we assayed the activity of this enzyme and determined immunologically the level of carbamoyl phosphate synthetase I in liver homogenates from young (4 months) and old (18 or 26 months) rats. In addition, we used electron microscopic immunogold procedures to locate and measure the amount of the enzyme in the mitochondrial matrix. There is no significant change in enzyme activity or enzyme protein content with age, although there is a higher concentration of the enzyme in the mitochondria (c. 1.5 times greater) from old rats, which is compensated by a decrease in the fractional volume of the mitochondrial compartment during aging.
Localization of cerium-based reaction products by scanning laser reflectance confocal microscopy.
Authors: Robinson J M , Batten B E
Published: April 2011

Scanning laser confocal microscopy was utilized to visualize sites of hydrogen peroxide release from stimulated neutrophils and lysosomal acid phosphatase in these and other cells using cerium in the detection systems. Imaging of the cerium-containing reactions was achieved by employing the reflectance mode of this instrument. Localization of these products at the light microscope level was direct and did not require other reactions to generate a visible product. This new approach to cerium cytochemistry should prove useful for many applications.
Detection of calcitonin-encoding mRNA by radioactive and non-radioactive in situ hybridization: improved colorimetric detection and cellular localization of mRNA in thyroid sections.
Authors: Denijn M , De Weger R A , Berends M J , Compier-Spies P I , Jansz H , Van Unnik J A , Lips C J
Published: April 2011

The localization of mRNA encoding calcitonin was studied by in situ hybridization using 35S-labeled RNA probes and biotin-labeled DNA probes. Radiolabeled probes were detected by autoradiography and biotin-labeled probes by streptavidin-biotin-peroxidase. To intensify the colorimetric signal, the indirect avidin-biotin complex (ABC) method was performed. However, the results were often variable. To improve the sensitivity, the peroxidase reaction signal was enhanced with a gold-silver deposit intensification reaction. To shorten the incubation times and to enhance the colorimetric reaction, several reaction steps were performed in a microwave oven. The localization of calcitonin mRNA in thyroid tissue, as detected with in situ hybridization, was confirmed by immunohistochemical localization of the calcitonin polypeptide. The results of in situ hybridization using biotinylated probes were compared to in situ hybridization using radioactive probes. Our data show that the results of in situ hybridization applied on frozen and paraffin-embedded sections using biotinylated DNA probes, detected with an indirect streptavidin-biotin-peroxidase reaction and intensified by silver-gold enhancement, were comparable to those obtained with radioactive probes. The localization of calcitonin encoding mRNA was in agreement with the localization of the calcitonin polypeptide.
Synthesis of Y chromosome-specific labeled DNA probes by in vitro DNA amplification.
Authors: Weier H U , Segraves R , Pinkel D , Gray J W
Published: April 2011

We describe the use of in vitro DNA amplification for production of double-stranded, biotin-labeled DNA probes. Specifically, a 124 BP DNA segment of the Y chromosome-specific 3.4 KB repeat was amplified in preparations of human genomic DNA using the polymerase chain reaction (PCR) and a thermostable DNA polymerase. The PCR products were amplified further in the presence of a molar excess of biotin-11-dUTP. The resulting double-stranded DNA segments showed a high amount of incorporated biotin-11-dUTP. The probes were used in DNA-DNA hybridization experiments without further purification. When DNA sequences flanking the target region are known, probe generation by enzymatic amplification offers a rapid and efficient alternative to molecular cloning and nick translation.
Simultaneous detection of different mRNA sequences coding for neuropeptide hormones by double in situ hybridization using FITC- and biotin-labeled oligonucleotides.
Authors: Dirks R W , van Gijlswijk R P , Tullis R H , Smit A B , van Minnen J , van der Ploeg M , Raap A K
Published: April 2011

Oligonucleotides labeled with FITC or biotin were applied for detection of specific mRNAs in microscopic preparations by in situ hybridization. The oligonucleotides were labeled with one FITC or biotin molecule at the 5' end or with a tail of biotin molecules at the 3' end. The target sequences were mRNAs coding for an ovulation hormone (CDCH) in the caudodorsal cells (CDC) of the pond snail Lymnaea stagnalis and a molluscan insulin-like peptide (MIP) in the light green cells (LGC) of the same organism. The hybridized oligonucleotides were detected either directly after the hybridization procedure by fluorescence microscopy or indirectly after an immunocytochemical procedure to visualize the biotin or FITC moiety. The results indicate that the detectability of the mRNA sequences is at least partially dependent on the accessibility of the target sequences for the immunocytochemical detection systems. The positive hybridization results obtained with oligonucleotides containing different labels enabled us to perform double hybridization experiments for simultaneous detection of CDCH and MIP mRNAs in one tissue section. Using FITC- and biotin-labeled oligonucleotides, we also demonstrated simultaneously different sequences on the same mRNA molecule.
Internalization of pulmonary surfactant into lamellar bodies of cultured rat pulmonary type II cells.
Authors: Kalina M , Socher R
Published: April 2011

We investigated the uptake of surfactant by isolated alveolar type II cells by using native pulmonary surfactant complexed with colloidal gold. Internalization to lamellar bodies (LB) occurred via vesicles (mainly coated) and the endosomal system. The highest labeling density was found in the endosomal system: vacuoles and the electron-lucent multivesicular bodies (MVB), which were labeled within 10 min. The labeling of electron-dense MVB (D-MVB) and LB was time dependent, reaching a plateau after 120 min, at which time approximately 30% and 70% of the LB and D-MVB were labeled, respectively. Internalization of surfactant-gold was inhibited by the addition of native surfactant or treatment of the gold complex with antibody against surfactant apoproteins. The internalization pathway of lectin from Macula pomifera (MPA) complexed with gold was compared to that of surfactant. Both pathways were found to be similar, except that mainly smooth vesicles rather than coated ones were involved in the process of MPA-G internalization. The partial labeling of the LB, the possible routing to lysosomes, and the endosomes as junction between the biosynthetic and endocytic pathways are discussed.
The endomembrane system of cholinergic and non-cholinergic neurons in the medial septal nucleus and vertical limb of the diagonal band of Broca: a cytochemical and immunocytochemical study.
Authors: Palacios G
Published: April 2011

Coronal vibratome sections of the rostral part of the medial septum (MS) and vertical limb of the diagonal band of Broca (VDB) nuclei were studied by an immunocytochemical technique using a monoclonal antibody against choline acetyltransferase (ChAT) and a double histochemical method for detection of acid phosphatase (AcPase) and nucleoside diphosphatase (NDPase) activity. The electron microscopic morphology of ChAT-immunoreactive and non-immunoreactive neurons was compared with similar neurons showing both AcPase and NDPase activity. ChAT-labeled and non-labeled neurons were well differentiated by the organization of the endomembrane system and especially by the structure of the rough endoplasmic reticulum (RER) and associated lamellar bodies. These results support the theory that the peculiar ultrastructure of the lamellar bodies in each neuron is related to the pattern of organization of the endomembrane system and its function. The significance of the lamellar bodies is discussed, and the data of the present work, together with findings described by other investigators. These data suggest that these bodies are predominant in efferent projection neurons in the basal forebrain nuclei.
Stability and localization of biotin-labeled murine monoclonal antibody to human gastric cancer in normal mice and nude mice-human tumor models.
Authors: Yokota T , Yamaguchi T , Kitamura K , Otsuji E , Sawai K , Takahashi T
Published: April 2011

We examined the tissue localization of biotin-labeled murine monoclonal antibody (MAb) S202 directed against the human scirrhous gastric carcinoma cell line MK-01 in normal and tumor-bearing mice after intravenous (IV) administration. The biotin-labeled MAb proved to be stable in vivo under normal conditions, antibody titer being 1:256 at 4 hr after IV injection. At 24 hr after injection, the tumor was stained by the avidin-biotin-peroxidase complex (ABC) method. Biotin-labeled MAb was found to be suitable for detection of the xenografted tumor of nude mice. This study provides new information concerning the dynamics of the distribution of biotin-labeled MAb in vivo.
Stereoscopic observation of enzyme distribution in lowicryl K4M-embedded specimens by conventional electron microscopy.
Authors: Tomiyasu K , Nasu F , Igarashi H , Inomata K
Published: April 2011

The present investigation was undertaken to explore the value of the Lowicryl K4M embedding technique for enzyme histochemical examination of semi-thin sections. The low-temperature embedding procedure with Lowicryl K4M was found to provide favorable conditions for preservation of enzyme activity in tissue samples. We tested the histological effects of various fixatives; the best results were obtained using 4% paraformaldehyde when testing for AcPase, AlPase, TPPase, and Mg-ATPase in the dorsal root ganglion. The three-dimensional cellular fine structure could be clearly seen in stereo pair pictures under stereoscopy.
Fluid-phase endocytosis by intrahepatic bile duct epithelial cells isolated from normal rat liver.
Authors: Ishii M , Vroman B , LaRusso N F
Published: April 2011

Although recent data from our laboratory have established the occurrence of receptor-mediated endocytosis in intrahepatic bile duct epithelial cells (IBDEC) isolated from normal rat liver, no studies have assessed the role of isolated IBDEC in fluid-phase endocytosis. Therefore, to determine if IBDEC participate in fluid-phase endocytosis, we incubated morphologically polar doublets of IBDEC isolated from normal rat liver with horseradish peroxidase (HRP, 5 mg/ml), a protein internalized by fluid-phase endocytosis, and determined its intracellular distribution by electron microscopic cytochemistry. Pulse-chase studies using quantitative morphometry were also performed to assess the fate of HRP after internalization. After incubation at 37 degrees C, IBDEC internalized HRP exclusively at the apical (i.e., luminal) domain of their plasma membrane; internalization was completely blocked at 4 degrees C. After internalization, HRP was seen in acid phosphatase-negative vesicles and in acid phosphatase-positive multivesicular bodies (i.e., secondary lysosomes). Small acid phosphatase-negative vesicles containing HRP moved progressively from the apical to the basal domain of IBDEC. Pulse-chase studies showed that HRP was then discharged by exocytosis at the basolateral cell surface. These results demonstrate that IBDEC prepared from normal rat liver participate in fluid-phase endocytosis. After internalization, HRP either is routed to secondary lysosomes or undergoes exocytosis after transcytosis from the luminal to the basolateral cell surface. Our results suggest that IBDEC modify the composition of bile by internalizing both biliary proteins and fluid via endocytic mechanisms.
Glycosaminoglycans of bovine aorta endothelial cells: identification and localization by use of a platelet factor 4-fluorescein probe.
Authors: Silbert D I , Gill P J , Humphries D E , Silbert J E , Culp L A , Silbert C K
Published: April 2011

We utilized platelet factor 4 (PF4) conjugated to fluorescein to stain the proteoglycans of permeabilized fixed bovine aorta endothelial cells in monolayer culture. Treatment of the monolayers with chondroitin ABC lyase and/or a preparation from Flavobacterium heparinum was used to remove chondroitin sulfate and/or heparan sulfate before staining, with resultant separate identification and partial localization of these glycosaminoglycans. When PF4-fluorescein was utilized with untreated control monolayers, fairly uniform reticular, perinuclear, and cell surface fluorescence was seen. After treatment with chondroitin ABC lyase, fluorescence was retained only on the cell surface. In contrast, treatment with the F. heparinum preparation resulted in the loss of all cell surface fluorescence. Use of both glycosaminoglycan lyases together resulted in loss of essentially all the fluorescence. The cell surface heparan sulfate observed by fluorescence after removal of cell surface chondroitin sulfate appeared to be unevenly distributed, with a heavier accumulation at one pole of each cell. This technique offers a specific method for identification and partial localization of cell surface heparan sulfate.
Monoclonal antibody (SBU-1 and SBU-3) identification of cells dissociated from the sheep placentomal trophoblast.
Authors: Lee C S , Ralph M M , Gogolin-Ewens K J , Brandon M R
Published: April 2011

We studied the localization of alpha-keratin in the sheep placenta using an alpha-keratin-specific monoclonal antibody (MAb) SBU-1, and examined the feasibility of using this MAb as a marker for determining the purity of isolated uninucleate cells from the placentomal trophoblast. At about 30-50 days of gestation the placentomal and interplacentomal uninucleate cells and some binucleate cells were stained by SBU-1, whereas only the apical region of the syncytial cytoplasm was stained with this MAb. Other cells stained included the uterine and endometrial glandular epithelial cells and fibroblast-like cells in the endometrium and chorionic villi. At about 100-130 days of gestation only the trophoblast uninucleate cells were stained by SBU-1. Approximately 60% of cells isolated from placentomes at 100-130 days of gestation were stained by SBU-1, and they had similar morphological features to the trophoblast uninucleate cells. The number of binucleate cells present was confirmed by their affinity for MAb SBU-3. These results show that MAb SBU-1 is an excellent marker for trophoblast uninucleate cells from placenta of sheep at the later stages of pregnancy.
Immunochemical and immunocytochemical studies of the C-propeptide of type II procollagen in chondrocytes of the growth plate.
Authors: Lee E R , Matsui Y , Poole A R
Published: April 2011

The C-propeptide of type II procollagen has previously been implicated in cartilage calcification. To further characterize this propeptide, we have investigated its molecular status and intracellular distribution in bovine fetal growth plate chondrocytes, particularly within the calcifying zone, using cell isolation, Western blotting, and localization with immunofluorescence and immunogold techniques. We found that in all cells freshly isolated by collagenase digestion the C-propeptide was a component of type II pro-alpha chains. No free C-propeptide was detected intracellularly. In situ localization of the C-propeptide by immunostaining employing immunofluorescence revealed the presence of procollagen in most growth plate cells, staining being most intense in hypertrophic cells. In the latter, large dilations of the rough endoplasmic reticulum were observed. These were not found in proliferating cells and had an approximate diameter of 5 microns. With immunogold localization these, together with Golgi-derived secretory granules, stained for the C-propeptide. These combined results suggest that in all cells of the growth plate the C-propeptide is a constituent part of type II collagen pro-alpha chains, and that it is usually segregated in the rough endoplasmic reticulum at a time when, according to other studies, collagen synthesis ceases in the lower hypertrophic zone and calcification of the extracellular matrix ensues. This suggests that the intracellular translocation of type II collagen pro-alpha chains may change in hypertrophic cells at this time.
Double-labeling of tissue containing the carbocyanine dye DiI for immunocytochemistry.
Authors: Elberger A J , Honig M G
Published: April 2011

The fluorescent carbocyanine dye DiI can be used for retrograde and anterograde labeling of neuronal pathways. To investigate the possible neurochemical identity of DiI-labeled neuronal cell bodies and terminals, we used a procedure for double-labeling of the same tissue with antisera to specific neuroactive substances. This procedure involves visualizing the immunohistochemical label with an FITC-conjugated secondary antiserum. Both labels can be viewed in the same tissue by fluorescence microscopy, and individual cell bodies and processes double-labeled with DiI and antiserum can be identified by switching between filter sets appropriate for rhodamine (to see the DiI labeling) and for fluorescein (to see the immunhistochemical labeling). The method has been used with primary antisera to excitatory and inhibitory amino acid neurotransmitters, as well as to neuropeptides, and is likely to be useful with antibodies against a wide variety of substances. Several other immunocytochemical methods were found to be incompatible with DiI labeling.
Alcohol abuse-related mesangial glomerulonephritis: immunoelectron microscopy.
Authors: Smith S M , Welford A , Chiovetti R , Hoy W E
Published: April 2011

Immunoelectron microscopy, using post-embedding immunohistochemistry with colloidal gold, was performed on renal samples from forensic autopsies. We confirmed that electron-dense deposits seen in alcohol abuse-related mesangial nephritis correspond to immunoglobulins, as has been shown previously by others in idiopathic cases. We investigated seven control samples and 13 specimens from individuals with evidence of alcohol abuse, six of whom had mesangial nephritis with IgA deposition. We found concentration of the gold particles over large electron-dense deposits in four of six cases of mesangial nephritis, confirming that they correspond to the IgA shown by immunofluorescence. Furthermore, a similar concentration of gold particles was not observed in control cases or in alcoholics without mesangial glomerulonephritis (4/6 vs 0/14; p = 0.005). IgM, seen as small aggregates, was confirmed in only two of six of the same cases. This is the first time that immunoelectron microscopy is performed on tissues obtained post mortem.