bluuebunny/crossref_metadata_2025 huggingface dataset:
Acute action of DSP-4 on central norepinephrine axons: biochemical and immunohistochemical evidence for differential effects.
Authors: Grzanna R , Berger U , Fritschy J M , Geffard M
Published: April 2011

Previous immunohistochemical studies of the long-term effects of the noradrenergic neurotoxin DSP-4 have demonstrated a remarkably selective vulnerability of norepinephrine (NE) axons of the locus coeruleus (LC). NE axons originating in non-LC NE neurons appear to be largely resistant to the neurotoxic action of DSP-4. We conducted this study to evaluate the acute effects of DSP-4 on NE axons in four different brain regions: cerebral cortex, cerebellum, ventral forebrain, and hypothalamus. NE levels were determined by high-performance liquid chromatography (HPLC) 6 and 24 hr and 14 days after DSP-4 administration. NE axons in these brain regions were visualized in brain sections at 6 and 24 hr after drug treatment, using a specific antiserum to NE. HPLC assays revealed profound reductions of NE levels in cerebral cortex and cerebellum, but only minor decreases in ventral forebrain and hypothalamus. NE immunohistochemistry showed dramatic differences in the acute effects of DSP-4 on NE axon staining: nearly complete loss of staining in cortex and cerebellum, in contrast to an almost unchanged staining pattern in ventral forebrain and hypothalamus. This study demonstrates that NE immunohistochemistry is a valuable tool to assess the acute effects of DSP-4 on NE axons in different brain regions. The results provide the first direct evidence that NE axons are not uniformly acted on by DSP-4 and suggest that the acute effects of DSP-4 are restricted to LC axons.
Electron microscopy of gold-labeled human and equine chromosomes.
Authors: Messier P E , Drouin R , Richer C L
Published: April 2011

We present an immunochemical technique for the detection of 5-bromo-2'-deoxyuridine (BrdU) incorporated discontinuously into the chromosomal DNA. A monoclonal anti-BrdU antibody and a protein A-gold complex were used to produce chromosome banding of human and equine chromosomes, specific for electron microscopy (EM). Well-defined bands, symmetry of sister chromatids, concordance between homologues, and band patterns similar to those observed by light microscopy facilitate chromosome identification and karyotyping. From prophase to late metaphase, chromosomes condense and bands appear to fuse. The fusion appears to be owing to chromatin reorganization. Our results underline the value of using immunogold reagents, which are ideal probes for antigen localization on chromosomes.
Cell kinetics of human tumors by in vitro bromodeoxyuridine labeling.
Authors: Meyer J S , Nauert J , Koehm S , Hughes J
Published: April 2011

We labeled active S-phase cells in primary breast carcinomas with a modified 5-bromo-2'-deoxyuridine (BrdU) procedure using a silver-enhanced colloidal gold visualization step. Separate samples of 29 tumors were labeled with BrdU or tritiated thymidine ([3H]-dThd), and the labeling indices (LI) from the two methods were equivalent (Spearman's correlation coefficient = 0.96). Three breast carcinomas were incubated in various mixes of both BrdU and [3H]-dThd and developed sequentially for each. Paired photomicrographs showed that the same nuclei were labeled by either precursor. The in vitro method yielded LIs similar to those reported after in vivo pulse BrdU labeling for tumors of the central nervous system. The BrdU LI correlated significantly (r = 0.76, p less than 0.001) with % S-phase by DNA flow cytometry in 33 breast carcinomas. The BrdU labeling method is simpler and more rapid than the [3H]-dThd procedure (1-2 days for completion for the former, 7-10 days for the latter), and it provides an equivalent measurement of proliferative index.
Characterization of nonspecific esterase activity in macrophages and intestinal epithelium of the rat.
Authors: Stadnyk A W , Befus A D , Gauldie J
Published: April 2011

We determined the histochemical characteristics of nonspecific esterase in different populations of rat macrophages. The cells included alveolar and peritoneal macrophages recovered by lavage and a mixed cell population obtained by collagenase digestion of the small intestine. The histochemically localized enzyme activity of alveolar and peritoneal macrophages was cytoplasmic, diffuse, and inhibited by sodium fluoride. Both populations were effectively stained using alpha-naphthyl acetate and alpha-naphthyl butyrate as the esterase substrate. When the intestinal cells were examined for activity, a greater percentage of cells showed positive nonspecific esterase than would be predicted by differential counts for macrophages on the basis of morphological criteria. We confirmed, using cell smears and tissue sections, that rat intestinal epithelial cells, a prominent component of the isolated cell population, possessed esterases that react similarly to macrophage esterases with histochemical procedures.
Brain-associated small-cell lung cancer antigen (BASCA) is expressed in developing lung: an immunohistochemical and immunoelectron microscopic study.
Authors: Fukayama M , Funata N , Hayashi Y , Maeda Y , Koike M , Watanabe J , Okabe T
Published: April 2011

Expression of brain-associated small-cell lung cancer antigen (BASCA) in developing lung and in lung tumors was investigated immunohistochemically and immunoelectron microscopically with monoclonal antibodies recognizing different epitopes of BASCA. In fetal lung, epithelial and mesenchymal cells had different spatial and temporal expression patterns, in contrast to the consistent pattern in neural cells. The cell membranes of epithelial cells of the proximal bronchial tubes were diffusely positive at the pseudoglandular stage. Ciliated cells lost immunoreactivity shortly after their emergence, but non-ciliated cells, including endocrine cells, lost it at the alveolar stage. The immunoreactivity in mesenchymal cells was reduced in the proximal airway, but positivity remained in the distal lung later during the postnatal period. All endocrine tumors of the lung, defined by diffuse synaptophysin immunoreactivity, expressed BASCA, but some non-endocrine carcinomas which also lacked densely cored granules ultrastructurally, showed BASCA positivity. The temporal and spatial pattern of BASCA expression in the developing lung suggests that BASCA plays an active role in lung morphogenesis. BASCA may be expressed as an oncofetal substance in some non-endocrine carcinomas of the lung.
A spectrophotometric method for determination of fluorophore-to-protein ratios in conjugates of the blue fluorophore 7-amino-4-methylcoumarin-3-acetic acid (AMCA)
Authors: Wessendorf M W , Tallaksen-Greene S J , Wohlhueter R M
Published: April 2011

7-Amino-4-methylcoumarin-3-acetic acid (AMCA) has been found to be a useful fluorophore for immunofluorescence. The present study describes a spectrophotometric method for determining the ratio of moles AMCA to moles protein (or the f/p ratio) in an AMCA-conjugated IgG. The concentration of a substance absorbing light can be determined spectrophotometrically using Beer's Law: Absorbance = Concentration x Extinction coefficient. From Beer's law, one can derive the following formula for determining the f/p ratio of AMCA-IgG conjugates: f/p = (epsilon 280IgG).A350 - (epsilon 350IgG).A280/(epsilon 350AMCA).A280 - (epsilon 280AMCA).A350 where A is the optical density of the conjugate at the given wavelength and epsilon is the extinction coefficient of a substance at the wavelength specified. Using conjugates of model proteins, it was found that the extinction coefficients of the AMCA moiety of AMCA-conjugated protein were 1.90 x 10(4) at 350 nm and 8.29 x 10(3) at 280 nm. Similarly, it was found that the extinction coefficients of swine IgG were 1.56 x 10(3) at 350 nm and 1.26 x 10(5) at 280 nm. Thus, for AMCA-conjugated swine IgG: f/p = (1.26 x 10(5)).A350 - (1.56 x 10(3)).A280/(1.47 x 10(4)).A280 - (6.42 x 10(3)).A350 [corrected]. Based on this formula, the f/p ratios of some AMCA-IgG conjugates useful for immunohistochemistry have been found to range between 6 and 24.
Light microscopic localization of glycosyltransferase activities in cells and tissues.
Authors: Benau D A , Schumacher W G , McGuire E J , Fitzpatrick-McElligott S , Storey B T , Roth S
Published: April 2011

We describe an assay for light microscopic visualization of specific glycosyltransferases on tissue sections or on cells. The assay uses a sequence of enzyme reactions that yields two moles of NADH for each mole of the uridine-5'-diphosphate (UDP) released during transfer of a monosaccharide from a UDP sugar to an acceptor. When diaphorase and tetrazolium salts are present in the incubation mixture, the tetrazolium salts are reduced to colored diformazans, which precipitate at the sites of glycosyltransferase activity. The validity of the assay was established by applying the technique to spermatozoa and liver, in which some glycosyltransferases have previously been localized. When suspensions of mouse spermatozoa were assayed for galactosyltransferase (GalTase) activity, diformazan precipitates appeared on the plasma membranes overlying the anterior heads of the spermatozoa, in agreement with immunochemical localizations. In mouse liver slices assayed with bilirubin as acceptor for glucuronyltransferase (GluTase) activity, dense diformazan deposits appeared on the hepatocytes but not on endothelial cells, also in agreement with immunochemical data. In the absence of acceptor or UDP sugar donor, diformazan deposits were minimal and random in all tissues tested. The assay's versatility was tested by incubating tissues with different sugar donors and acceptors to localize other sites of transferase activity. In mouse frozen liver sections, GalTase activity occurred in both hepatocytes and endothelial cells; in sections of rat submaxillary glands, GalTase activity was detected in mast cells. In liver sections, GlcuTase activity with o-aminophenol as acceptor was located primarily on the endothelial cells. With the appropriate sugar donor and acceptor, this assay should detect any transferase, other than the glucosyltransferases, that utilizes UDP sugars.
Immunocytochemical demonstration of vasopressin binding in rat kidney.
Authors: Fadool J M , Aggarwal S K
Published: April 2011

We investigated the immunoperoxidase demonstration of vasopressin (VSP) bound to paraffin-embedded sections of rat kidney and the effects of various fixatives. Slices of rat kidney from normal and 4-day water-deprived rats were incubated with 10(-7) M VSP, fixed, and embedded in paraffin. Hydrated sections of these tissues were again incubated with 10(-7) M VSP or 10(-7) M VSP and 10(-5) M oxytocin (OXY). VSP bound to the sections was demonstrated using rabbit anti-Arg8 VSP antiserum and peroxidase-labeled second antibody. In sections of kidney from both normal and water-deprived rats, immunoperoxidase labeling was most intense in the renal papilla and was restricted to the cells of the ducts of Bellini and loops of Henle. In the medulla, the collecting ducts and medullary thick ascending limbs of Henle were moderately stained. In the normal kidney sections there was no staining of the proximal tubules, distal convoluted tubules (DCT), and only slight staining of the cortical collecting ducts (CCD). However, in the water-deprived rats there was a considerable increase in the staining of the DCT and CCD. Simultaneous incubation in OXY and VSP resulted in reduced immunoperoxidase labeling of the tubules. Omission of VSP incubation led to a similar decrease in stain intensity, indicating a specificity for the sites of VSP binding. This technique allows the identification of cells responsible for the binding of VSP in the kidney.
Cytochemical localization of calcium and Ca2+,Mg2(+)-adenosine triphosphatase in colchicine-altered rat incisor ameloblasts.
Authors: Eisenmann D R , Salama A H , Zaki A M , Ashrafi S H
Published: April 2011

Colchicine is known to affect secretory, transport, and degradative functions of ameloblasts. The effects of colchicine on membrane-associated calcium and Ca2+,Mg2(+)-ATPase in secretory and maturation ameloblasts were investigated cytochemically. The pyroantimonate (PPA) method was used for localizing calcium and a modified Wachstein-Meisel medium was used to localize Ca2+,Mg2(+)-ATPase. Sections representing secretory and early maturation stages were examined by transmission electron microscopy. Morphological changes induced by colchicine included dislocated organelles and other well-established reactions to such anti-microtubule drugs. Calcium pyroantimonate (Ca-PA) deposits in most ameloblast types were markedly reduced, with the greater reduction occurring in those cells more severely altered morphologically. However, the cell membranes of both control and experimental smooth-ended maturation ameloblasts were essentially devoid of Ca-PA. The normal distribution and intensity of Ca2+,Mg2(+)-ATPase was not affected by colchicine. Because the observed reduction of membrane-associated calcium is apparently not mediated by Ca2+,Mg2(+)-ATPase in this case, other aspects of the calcium regulating system of ameloblasts are apparently targeted by colchicine.
Calcitonin, prostaglandin E2, and dibutyryl cyclic adenosine 3',5'-monophosphate disperse the specific microfilament structure in resorbing osteoclasts.
Authors: Lakkakorpi P T , Väänänen H K
Published: April 2011

Bone resorbing osteoclasts form a specific microfilament structure at the attachment area, in which vinculin and talin appear as a double-circle structure and F-actin fills the space between these circles. This distribution of microfilaments is associated with the resorption lacunae, and F-actin, vinculin, and talin zones correspond roughly to the edges of the lacunae. In the present work, we examined by immunofluorescence the effects of calcitonin (CT) and prostaglandin E2 (PGE2), inhibitors of osteoclastic activity, as well as dibutyryl cyclic AMP (Bt2cAMP) and cytochalasin B, on the microfilament organization in resorbing osteoclasts. CT, PGE2, and Bt2cAMP rapidly dispersed the specific microfilament structure in resorbing osteoclasts. All microfilament proteins studied (vinculin, talin, and F-actin) spread to the central areas of the original circles. The effect of CT was dose dependent. The effects of CT and PGE2 could be reversed, but recovery was slower after CT treatment than after PGE2 treatment. Cytochalasin B entirely destroyed the F-actin organization but only partially the vinculin organization. The results suggest that one structural change leading to the inactivation of the osteoclasts caused by CT and PGE2 is the disintegration of the microfilament structure at the attachment area of resorbing osteoclasts.
Detection of sialic acid residues in the axonal reticulum of rat superior cervical ganglion cells by lectin-gold cytochemistry.
Authors: Quatacker J R , Annaert W G , De Potter W P
Published: April 2011

Highly glycosylated compounds have been demonstrated in the axonal reticulum elements of the superior cervical ganglion cells of the rat, and this is considered to suggest a connection of the reticulum with the trans Golgi side. In the present study, the axonal reticulum and the Golgi elements were further characterized by post-embedding methods of lectin-gold cytochemistry to determine their carbohydrate residues and to see, more specifically, if sialic acid residues could be detected in the axonal reticulum elements. Therefore, the affinity of neuronal cell structures for Limax flavus agglutinin (LFA), wheat germ agglutinin (WGA), and Ricinus communis agglutinin I (RCA-I) was tested in ultra-thin sections of glycolmethacrylate-embedded material, counterstained with phosphotungstic acid (PTA) at low pH. The trans Golgi network, the Golgi-associated axonal reticulum, the reticulum within axons, the large dense-cored vesicles, and the plasma membranes were reactive for all three lectins used. We conclude that the axonal reticulum elements carry sialic acid residues, relating them to the trans Golgi network. The present results support the concept that the axonal reticulum is an extension of the trans network of the Golgi apparatus specialized for neurosecretion.
A combined AgNOR-Feulgen staining technique.
Authors: Foucrier J , Rigaut J P , Pechinot D
Published: April 2011

We describe a new staining technique (H-Ag-S) which allows observation and counting of active nucleolus organizer regions (NORs) and evaluation of the amount of DNA in the same cell nucleus. The procedure consists of combining a modified AgNOR staining method with the Feulgen reaction. A sequential procedure is proposed, based on the determination of optimal staining conditions. The technique, which was designed to allow studies of correlations between the transcriptional activity of rDNA genes and the cell ploidy, was primarily developed for rat liver smears. It should be applicable to most biological preparations, but the optimal conditions might be variable.
Immunolabeling efficiency of protein A-gold complexes.
Authors: Ghitescu L , Bendayan M
Published: April 2011

A systematic study of the adsorption of protein A on colloidal gold particles varying in size from 5-16 nm was performed at different protein concentrations. The number of protein A molecules bound per colloidal particle was evaluated and the Scatchard analysis of the adsorption parameters was applied for each size of the colloid. The binding of protein A to the colloidal gold surface exhibited the same affinity pattern for all of the particle sizes. At low concentrations of stabilizing protein, adsorption took place with high affinity (Kd 1.96-3.3 nM) and the maximum number of protein A molecules attached with this affinity correlated well with the surface of the particle. At higher concentrations of protein A, adsorption exhibited a significantly lower affinity (Kd 530-800 nM), and no saturation was recorded. Competition by albumin did not reveal a preferential removal of the "low-affinity" bound protein A molecules, contradicting the model of successive shells of stabilizing protein around the colloidal particle. The immunolabeling efficiency of conjugates having the same size of gold nucleus but carrying different numbers of protein A molecules was comparatively investigated by quantitative post-embedding immunocytochemistry. Protein A-gold formed with 5-10-nm colloids gave the highest intensity of labeling when carrying the maximum number of protein A molecules that could be adsorbed with high affinity. Overloading as well as underloading these complexes resulted in a significant decrease of their immunoreactivity. The most efficient conjugates were obtained when stabilization was performed with 6 micrograms protein A/ml gold sol of 5 and 10 nm particle diameter, and 15 micrograms protein/ml of 15-nm colloid.
Microphotometric analysis of cytochrome P-450 in periportal, midzonal, and perivenular hepatocytes of mice treated with phenobarbital.
Authors: Kanai K , Watanabe J , Kanamura S
Published: April 2011

To obtain detailed information on the increase of cytochrome P-450 (P-450) content in periportal, midzonal, and perivenular hepatocytes after phenobarbital (PB) administration, and to study the mechanism of increased P-450 in the endoplasmic reticulum (ER), we estimated microphotometrically the P-450 content and morphometrically the area of ER in hepatocytes of three zones from mice injected with 35, 50, 100, or 150 mg/kg of PB for 3 days. The amount of P-450 per unit cytoplasmic volume and the number of P-450 molecules per unit ER area (P-450 number) were increased by injection of 50, 100, or 150 mg/kg, and the ER area per unit cytoplasmic volume was increased by injection of 100 or 150 mg/kg, in hepatocytes from all three zones. Thus, the amount of P-450 in hepatocytes appeared in general to increase multiplicatively by simultaneous increases in both the P-450 number and the ER area. Furthermore, we could recognize two general types of relationship in the P-450 number and ER area between the patterns of change and the increasing doses: (a) increase in the P-450 number without ER proliferation (active type) in periportal and perivenular hepatocytes after injection of low doses; and (b) increase in ER proliferation without increase in the P-450 number (passive type) in hepatocytes of all three zones after injection of high doses.
An improved staining method for rat microglial cells using the lectin from Griffonia simplicifolia (GSA I-B4).
Authors: Streit W J
Published: April 2011

A simple method for the lectin histochemical visualization of rat microglial cells is described. Advantages include ease of fixation of brain tissue using paraformaldehyde, and rapidity of tissue processing by vibratome sectioning. Furthermore, in addition to providing good structural preservation, the method achieves improved lectin binding, resulting in complete labeling of all microglial cells and in superior visualization of cellular processes. The lectin histochemical technique for rat microglia has the potential to be adapted to any mammalian species, and should prove valuable for neuroscientists interested in studying this glial cell type.
Effect of resin use in the post-embedding procedure on immunoelectron microscopy of membranous antigens, with special reference to sensitivity.
Authors: Shida H , Ohga R
Published: April 2011

To investigate quantitatively the effect of resins on the sensitivity of immunoelectron microscopy of membranous antigen, ultra-thin sections of bovine epithelial tissue embedded in five different kinds of resins [JB-4 (JB4), LR Gold (LRG), Lowicryl K4M (K4M), Quetol 812 (Q812), and Spurr's (Spurr) resin] were labeled specifically with anti-desmosomal glycoprotein I(DGI) antibody followed by protein A-gold (PAG) conjugates. When we compared the labeling intensity expressed as the number of PAG particles per 500-nm length of the desmosomal region along the membrane, three hydrophilic resins (JB4, LRG, and K4M) showed much greater levels of labeling intensity than did epoxy resins (Q812 and Spurr), which had a negative value. The three hydrophilic resins showed only minor differences in their levels of labeling intensity. The intensity obtained with JB4, which was the highest of the three, was further increased by pretreatment of the ultra-thin sections with methyl methacrylate monomer (MM) for 5 min. On the basis of these results, wide applicability of this new technique for membranous antigens, which have been difficult to detect positively by any previously employed techniques, is suggested.
A high-resolution tungstate membrane label.
Authors: Hainfeld J F , Lipka J J , Quaite F E
Published: April 2011

A new class of membrane labels was synthesized which contain a tungstate cluster (having 11 tungsten atoms) and an aliphatic organo-tin moiety with various chain lengths (C4, C8, C12, C18, C22). These molecules were found to insert into synthetic phospholipid vesicles and biological membranes (human red blood cell membranes). The tungstate clusters can be individually visualized in the high resolution STEM or seen en mass in thin-sectioned labeled membranes in the CTEM. These new labels should provide a means for direct high-resolution imaging of lipid-phase systems.
Alkaline phosphatase and dipeptidylpeptidase IV staining of tissue components of skeletal muscle: a comparative study.
Authors: Grim M , Carlson B M
Published: April 2011

A combined alkaline phosphatase (AP) and dipeptidlypeptidase IV (DPP IV) staining reaction has demonstrated enzymatic heterogeneity of the arterial and venous segments of capillaries in rat skeletal muscle. This study compared the staining reactions of skeletal muscles in many commonly used laboratory animals, including the axolotl, chick, quail, Monodelphys, rat, mouse, hamster, guinea pig, rabbit, dog, monkey, and human. DPP IV activity was found in the venous ends of the capillaries and in the endothelium of some larger veins in many of the species but was never demonstrated in the arterial side of the circulation. AP was found in the arterial ends of capillaries in all species except the axolotl, and it was also found in the endothelium of larger arteries of most species. AP activity was absent in venous endothelium of all species except for birds and Monodelphys. DPP IV activity was found in the perineurium of intramuscular nerves of most species, and AP activity was commonly seen in tendons and intramuscular connective tissue. The interspecies variability found in this study shows that care must be taken in comparing experimental data involving this technique from one species to another, but within a species the technique allows a fine level of discrimination between functionally distinct compounds of skeletal muscle tissue.
Immunogold electron microscopic demonstration of glutamate and GABA in normal and deafferented cerebellar cortex: correlation between transmitter content and synaptic vesicle size.
Authors: Hámori J , Takács J , Petrusz P
Published: April 2011

Selective labeling of mossy fiber terminals and parallel fibers was obtained in rat cerebellar cortex by a glutamate antibody produced and characterized by Hepler et al. The high-resolution electron microscopic immunogold demonstration of this amino acid offered the possibility of determining the size and shape of synaptic vesicles in glutamate-positive mossy endings. Mossy terminals that stained with the glutamate antibody formed two distinct populations, one with spherical synaptic vesicles with an average diameter of 34.0 nm (more than 90% of all mossy fiber endings) and one with pleomorphic and smaller synaptic vesicles which had an average diameter of 28.5 nm. We present experimental evidence that the mossy terminals with large round vesicles are of extracerebellar origin, whereas those with small pleomorphic synaptic vesicles are endings of nucleocortical fibers. The presence of two distinct classes of gamma-aminobutyric acid (GABA)-containing axon terminals within cerebellar glomeruli has also been demonstrated; those originating from the cerebellar nuclei contain large (36.2 nm) synaptic vesicles, whereas the majority of GABA-stained axon terminals that are of local (cortical) origin contain small (29.1 nm) synaptic vesicles. It therefore appears that, at least in the case of glutamate and GABA, morphological characterization of the axon terminals based on the size and shape of synaptic vesicles is not a reliable indicator of their functional nature (i.e., whether they are excitatory or inhibitory); convincing evidence for the identity of the transmitter can be obtained only by electron microscopic immunostaining procedures. Our results also suggest the existence of both inhibitory and excitatory feedback from cerebellar nuclei to cerebellar cortex.
c-erbB-2 expression in breast cancer detected by immunoblotting and immunohistochemistry.
Authors: Kerns B J , Pence J C , Huper G , Kinney R B , Iglehart J D
Published: April 2011

Evidence that the c-erbB-2 proto-oncogene is important in prognosis and oncogenesis in a number of human malignancies is increasing. DNA (Southern) hybridization and immunoblotting (Western) techniques are most commonly utilized to determine the amplification and protein expression of this proto-oncogene, respectively. These extraction techniques are often time consuming, costly, and subject to variability depending on the histological characteristics of the tumor. Immunohistochemistry (IHC), on the other hand, is more often time and cost effective. In addition, IHC may offer enhanced sensitivity over extraction techniques because of the in situ nature of analysis. In data presented here, 71 cases of human mammary carcinoma were concomitantly assessed for c-erbB-2 gene copy number and oncoprotein expression by dilution DNA hybridization and IHC, respectively. In 65 (92%) of 71 cases, high-level expression was associated with gene amplification, whereas moderate or low-level expression was associated with a normal diploid gene copy number. In five of the six discrepant cases, IHC predicted amplification which was not corroborated by Southern analysis. In these cases, tumor mass was limited by the intraductal component of the lesion or by an abundance of stromal elements within the specimen. In 39 of the 71 total cases, Western immunoblotting was compared with IHC in the assessment of oncoprotein expression. Concordance was found in 33 (85%) of 39 cases. In four of the six discrepant cases, high levels of c-erbB-2 expression were demonstrated by IHC but not by immunoblotting. In these cases, intraductal disease and stroma-rich tumors again led to a relative paucity of neoplastic tissue within the specimens. We conclude that IHC offers a favorable alternative to either Southern analysis or Western immunoblotting in the assessment of c-erbB-2 gene copy number and expression levels of oncoprotein in human mammary carcinoma. Furthermore, IHC may prove advantageous to either extraction technique in specimens with limited tumor mass, such as biopsy materials, stroma-rich tumors, or early stage lesions such as intraductal carcinoma.
Optimizing parameters for correlative immunogold localization by video-enhanced light microscopy, high-voltage transmission electron microscopy, and field emission scanning electron microscopy.
Authors: Simmons S R , Pawley J B , Albrecht R M
Published: April 2011

Correlative video-enhanced light microscopy, high-voltage transmission electron microscopy, and low-voltage high resolution scanning electron microscopy were used to examine the binding of colloidal gold-labeled fibrinogen to platelet surfaces. Optimal conditions for the detection of large (18 nm) and small (3 nm) gold particles are described.
Cellular distribution of prothymosin alpha and parathymosin in rat thymus and spleen.
Authors: Roson E , Garcia-Caballero G , Heimer E P , Felix A M , Dominguez F
Published: April 2011

By means of immunohistochemical methods, we have investigated the cellular distribution of prothymosin alpha and parathymosin in rat thymus and spleen, using specific antibodies raised against thymosin alpha-1 and against parathymosin. We observed prothymosin alpha immunoreactivity in lymphoid cells both in thymus and spleen. In the thymus, prothymosin alpha staining was more marked in cortex than in medulla. In the spleen, prothymosin alpha was found in lymphocytes of the periarteriolar lymphatic sheaths and was especially prominent in the germinal centers. Parathymosin immunoreactivity in the thymus was mainly localized in the medulla; positive cells were reticuloepithelial cells from the thymic reticulum and the blood barrier. Thymocytes were negative. In spleen, parathymosin was found in reticular cells arranged in a ring between the periarteriolar lymphatic sheath and the marginal zone. Our results do not support an exclusive role for these peptides as immune system hormones or cytokines.
Production of monoclonal antibodies against principal cells of the renal collecting duct by in vitro immunization with unsolubilized antigens.
Authors: Gilbert P , Mundel P , Minuth W W
Published: April 2011

We report the production of monoclonal antibodies (MAb) by an in vitro technique which react with principal cells of the renal collecting duct. Spleen cells were directly simulated in vitro with unsolubilized antigens, i.e., by direct contact with the apical site of cultivated principal cells or by contact with cell fragments. Out of several others two antibodies, IV1 and IV2, were selected, which specifically reacted with the principal cells of the collecting duct. MAbIV1 also reacted with Type A intercalated cells, indicating the existence of a common antigen in the apical membrane of both cell types. Type B intercalated cells were consistently unreactive. All other parts of the uriniferous tubule were also unreactive. In Western blot analysis MAb IV1 showed immunoreactivity with a 40 KD and a 43 KD antigen. Our experiments demonstrate the possibility of producing antibodies against unsolubilized antigens by a simple in vitro technique. The activity of particular lymphocyte in this in vitro system is shown by the specificity of the antibodies.
Immunohistochemical localization of phosphoenolpyruvate carboxykinase in adult and developing mouse tissues.
Authors: Zimmer D B , Magnuson M A
Published: April 2011

We used immunohistochemical techniques to analyze the cell distribution of phosphoenolpyruvate carboxykinase (PEPCK) in adult and developing mouse tissues. PEPCK immunoreactivity was detected in many tissues, including some that had not been previously reported to contain PEPCK enzyme activity (bladder, stomach, ovary, vagina, parotid gland, submaxillary gland, and eye). In some multicellular tissues, PEPCK immunoreactivity was observed in multiple cell types. Several tissues (spleen, thyroid, and submaxillary gland) contained no detectable PEPCK immunoreactivity. During development, PEPCK immunoreactivity was associated with the developing nervous system and somites in 15-day embryos. At prenatal day 18, PEPCK immunoreactivity was detected only in the nervous system. At prenatal day 20, PEPCK immunoreactivity was observed in many of the tissues that contain PEPCK in the adult, with the exception of liver, lung, and stomach. PEPCK immunoreactivity was detected in liver at postnatal day 1, lung at postnatal day 7, and stomach after postnatal day 21. The only tissue in which PEPCK immunoreactivity decreased during development was the pancreas, where PEPCK immunoreactivity was detected at prenatal day 20 and was present until postnatal day 21. These results suggest that PEPCK expression is cell-type specific, more widespread than previously thought, and differentially expressed during development.
Localization of pulmonary carbonyl reductase in guinea pig and mouse: enzyme histochemical and immunohistochemical studies.
Authors: Matsuura K , Hara A , Sawada H , Bunai Y , Ohya I
Published: April 2011

We studied the localization of carbonyl reductase (E.C. 1.1.1.184) in guinea pig and mouse lung by enzyme histochemistry and immunohistochemistry, using antibodies against the guinea pig lung enzyme which crossreacted with the lung enzymes of both animals. Carbonyl reductase activity was detectable in the bronchiolar epithelial cells of small airways and in alveolar cells. In the immunohistochemical staining for carbonyl reductase, the reaction was strongest in the non-ciliated bronchiolar cells (Clara cells) and was weak in the ciliated cells and type II alveolar pneumocytes. Injection of a single dose of naphthalene led to significant impairment of carbonyl reductase activity and of microsomal mixed-function oxidase activities in mouse lung, with a marked decrease in both activity and immunoreactive staining in the bronchiolar epithelial cells. The results indicate that carbonyl reductase is localized primarily in the Clara cells, which are known to be sites of pulmonary drug metabolism.
Immunogold-silver staining of mesangial antigen in lowicryl K4M- and LR gold-embedded renal tissue using epipolarization microscopy.
Authors: Shires M , Goode N P , Crellin D M , Davison A M
Published: April 2011

We examined the localization of a glomerular mesangial antigen with a Thy-1.1 monoclonal antibody by epipolarization microscopy (EPI) of silver-enhanced, immunogold-stained renal tissue embedded in LR Gold and Lowicryl K4M, and compared the attributes of these hydrophilic resins. Antigen was well preserved in tissue embedded in both resins. LR Gold-embedded tissue demonstrated excellent immunostaining properties, sectioned more easily, and showed better durability during staining than K4M. Lowicryl K4M-embedded tissue, however, displayed a phenomenon of self-illumination when counterstained with eosin which was not seen with LR Gold. This enabled immunostaining to be precisely related to tissue morphology without the necessity of simultaneous transillumination, which can be problematic when used in combination with EPI because of reflection of incident illumination from sub-stage optical surfaces.
Computerized quantification of immunofluorescence-labeled axon terminals and analysis of co-localization of neurochemicals in axon terminals with a confocal scanning laser microscope.
Authors: Mossberg K , Arvidsson U , Ulfhake B
Published: April 2011

The confocal scanning laser microscope (CSLM) offers improved optical resolution and contrast, high photometric precision, and the ability to make optical sections. These benefits were explored for use in quantitative analysis of immunofluorescence-labeled axon terminals. Guidelines were obtained for adjustments of the CSLM parameters. In the present applications, bleaching of the fluorescence did not represent a serious obstacle to analysis with the CSLM. A method was developed to distinguish the background fluorescence from the specific fluorescence labeling. This procedure made way for the development of automated quantification of immunolabeled axon terminals. The automated procedures substantially reduced the man-hour expenditure for analysis and provided highly reproducible quantifications compared with manual methods. The increased resolution and contrast of the CSLM allowed measurements of the fluorescence signal strength of individual axon terminals. The CSLM also allowed detection of co-localized neurochemicals in axon terminals.
Immuno- and affinity probes for electron microscopy: a review of labeling and preparation techniques.
Authors: Stirling J W
Published: April 2011

Immuno- and affinity probes are widely used in biology and medicine, and are becoming essential tools for the elucidation of cell structure and function. This article reviews and discusses the bewildering array of probes and preparation techniques now available for the investigation of sectioned material by transmission electron microscopy, with critical analysis of their merits. Emphasis is placed on immunogold probes and methods useful for routine preparation, gathering together information that may be used to improve labeling techniques. New data on inert dehydration for the localization of sensitive epitopes without chemical or cryofixation is presented.
A reliable method for simultaneous demonstration of two antigens using a novel combination of immunogold-silver staining and immunoenzymatic labeling.
Authors: Gillitzer R , Berger R , Moll H
Published: April 2011

We have developed a reliable and sensitive immunohistochemical staining technique which allows the simultaneous demonstration of two different antigens expressed in or on the same cell (referred to as mixed labeling), together with the evaluation of the general histopathological appearance of the tissue. The staining procedure combines a three-step (streptavidin-biotin) immunogold-silver staining (IGSS) with a three-step immunoenzymatic labeling. For this purpose, we investigated the compatibility of IGSS with various substrates of peroxidase or alkaline phosphatase (AP). Highly reliable and discernible mixed labeling was achieved only after initial labeling with IGSS followed by AP labeling using the substrates naphthol AS-MX phosphate/Fast Blue or naphthol AS-BI phosphate/New Fuchsin, respectively. To ensure utmost specificity, we applied FITC-conjugated mouse monoclonal antibodies and rabbit anti-FITC immunoglobulins visualized by AP-labeled immunoglobulins and the respective substrate in a final step. This novel approach provides an excellent means for demonstration of immunocompetent cells and unequivocal determination of the percentage of specific cell subsets in infiltrated tissue. The advantages of this method, as compared with double immunofluorescence or double immunoenzymatic labeling, were investigated and are discussed.
Improved immunocytochemical staining of carcinogen-DNA adducts by a capillary slot block system.
Authors: Scherer E , Van Benthem J
Published: April 2011

We developed an immunocytochemical protocol in which incubation occurs in a capillary slot instead of the conventional horizontal drop. Slots of constant width were formed by placing slides on top of each other with parafilm spacer layers in between. Cryostat or semi-thin plastic-embedded sections were cut from organs of carcinogen-treated experimental animals. Carcinogen-DNA adducts were visualized in the affected nuclei by a double peroxidase-antiperoxidase method using rabbit antisera specific for certain DNA adducts formed. The staining in capillary slot blocks offered better staining reproducibility than the conventional method. This is particularly important when the staining intensity must be quantified. In addition, handling of the blocks was substantially less laborious than the individual treatment of slides, making this protocol especially suitable for larger series of slides. Other applications for the capillary slot block protocol should be enzyme histochemistry and in situ hybridization.
Effects of bacterial lipopolysaccharide and calmodulin on Ca2(+)-ATPase and calcium in human natural killer cells, studied by a combined technique of immunoelectron microscopy and ultracytochemistry.
Authors: Kang Y H
Published: April 2011

Our previous studies indicate that bacterial lipopolysaccharide (LPS) enhances natural killer (NK) cell-mediated cytotoxicity and increases intracellular calcium (Ca2+) in hepatocytes. Calmodulin (CAM) regulates Ca2(+)-ATPase activity, intracellular Ca2+, and is also implicated in NK cell-mediated cytolysis. In the present work, the effects of LPS and CAM on Ca2(+)-ATPase and intracellular Ca2+ in human NK cells were studied by a combined technique of immunogold electron microscopy and ultracytochemistry. Peripheral blood mononuclear cells were treated with 100 micrograms/ml E. coli (0111:B4) LPS and/or 5 micrograms/ml CAM in RPMI 1640 medium at 37 degrees C for 1 or 4 hr. NK cells labeled with monoclonal anti-Leu-11a (CD16) antibody and colloidal gold-conjugated anti-mouse IgG were processed for cytochemical localization of Ca2(+)-ATPase and Ca2+. Ca2(+)-ATPase was localized in the plasma membrane of NK cells, and its activity was suppressed by LPS but was enhanced by CAM. However, no apparent changes in the enzyme reaction were observed when cells were exposed to CAM concomitantly with LPS or stimulated with LPS before CAM. Apparent reduction of the enzyme reaction was observed when LPS stimulation was preceded by CAM. Ca2(+)-ATPase reaction in mitochondria was observed only in NK cells exposed to CAM. Computer image analysis showed no changes in the intracellular Ca2+ in NK cells treated with LPS for 1 hr, whereas a significant increase in Ca2+ was found in cells exposed to LPS for 4 hr. The intracellular Ca2+ significantly decreased in NK cells treated with CAM or with a combination of LPS and CAM as compared to that of controls (p less than 0.05). The results indicate that CAM is capable of blocking or reversing the inhibitory effect of LPS on Ca2(+)-ATPase, and suggest that in human NK cells the plasma membrane-associated Ca2(+)-ATPase is responsible for extrusion of intracellular Ca2+.
Affinity cytochemical labeling of glomerular basement membrane anionic sites using specific biotinylation and colloidal gold probes.
Authors: Bertolatus J A
Published: April 2011

To label specific anionic sites on glomerular capillary wall structures, biotin was covalently linked to sialic acid residues by sequential treatment with mild peroxidation and biotin hydrazide, while carboxyl groups were biotinylated by exposure to the combination of biotin hydrazide and a water-soluble carbodiimide reagent. Optimal specific labeling of rat glomerular structures was obtained with in situ perfusion of the biotinylation reagents, followed by fixation in 4% phosphate-buffered paraformaldehyde, embedment in LR White resin, and post-embedment detection of biotinylated sites using a sequence of anti-biotin antibodies followed by a secondary antibody-colloidal gold conjugate. Attempts to use streptavidin-gold conjugates were not successful. Specificity of labeling was confirmed by enzymatic (neuraminidase) pre-treatment or by modification of carboxyl groups, as evaluated by electron microscopy and by solid-phase assays of solubilized glomerular basement membrane (GBM) components. In two rats with heavy proteinuria induced by doxorubicin (Adriamycin), a marked reduction in sialic acid residues within the GBM and on the epithelial cell surfaces was found, suggesting that reduced charge density attributable to abnormal sialylation may be important in the pathogenesis of nephrotic proteinuria.
Double immunocytochemical staining for in vivo detection of epitope specificity and isotype of antibody-forming cells against synthetic peptides homologous to human immunodeficiency virus-1.
Authors: Laman J D , Gerritse K , Fasbender M , Boersma W J , van Rooijen N , Claassen E
Published: April 2011

Many infections evoke a strong humoral immune response. Some (e.g., HIV-1, EBV, CMV) also lead to disorders of the B-cell system. Data concerning cell dysfunction are largely derived from in vitro studies, which necessarily exclude all microenvironmental influences. The aim of this study was to develop a tool for the investigation of epitope specific humoral immune responses in vivo. Mice were immunized with one of two synthetic peptides, both 21 amino acids long and homologous to regions of the HIV-1 gp160. Cryostat sections of spleen and lymph nodes were incubated with the corresponding peptide coupled to alkaline phosphatase and simultaneously incubated with peroxidase-conjugated rabbit antisera specific for mouse immunoglobulin isotypes. We were able to show simultaneous detection of epitope specificity, isotype, and localization of antibody-forming cells and immune complexes in tissue sections. It should prove useful for in vivo investigation of the development of specific (e.g., anti-HIV-1) humoral immune response, the determination of B-cell specificity in lymph node infiltrates, and the role of immune complexes in lymph node pathology.
Histoautoradiographic study of rRNA and mRNA transcription activity related to secretory function in lizard epididymis.
Authors: Faure J , Chevalier M , Mesure M , Dufaure J P
Published: April 2011

The lizard epididymis is an androgen-dependent organ whose epithelial cells undergo marked changes in structure and secretory activity during the annual cycle. These changes are connected to fluctuations of testosterone levels. During the breeding season, the epididymis produces a major protein secretion, the L-proteins. In the present work we studied the fluctuations of RNA synthesis and accumulation during the annual cycle by means of histoautoradiographic methods. Total RNA synthesis was determined by uridine incorporation; accumulation of rRNAs and L-protein mRNAs were determined by in situ hybridization. Total RNA synthesis began during reorganization (Phase I), then increased gradually during differentiation and growth (Phase II). The synthesis peaked during maturation (Phase III), but stopped abruptly during hypersecretory activity (Phase IV). The rRNAs were very abundant from Phase II to Phase IV, which is related to the presence of many ribosomes as revealed by electron microscopy. The mRNAs of L-proteins were detected only during Phases III and IV in all epithelial cells. For every phase of the sexual cycle there exists a strong correspondence between the changes in transcriptional activity (rRNA and specific mRNA) of the epithelial cells and changes in the testosterone levels.