bluuebunny/crossref_metadata_2025 huggingface dataset:
Plasminogen activating enzyme in cultured glioblastoma cells. An immunofluorescence study with monoclonal antibody.
Authors: Danø K , Dabelsteen E , Nielsen L S , Kaltoft K , Wilson E L , Zeuthen J
Published: April 2011

A monoclonal antibody against a 52,000 dalton human plasminogen activating enzyme (HPA52) was used for immunofluorescence staining of cultured glioblastoma cells. The fluorescence was located in the cytoplasm of the cells. A pronounced variation in the staining intensity was observed between the individual cells. The specificity of the fluorescent stain was supported by the findings that 1) no staining was obtained with a monoclonal antibody of the same subclass, but with irrelevant specificity (anti-2,4,6-trinitrophenyl); 2) adsorption with HPA52 purified to homogeneity removed the ability of anti-HPA52 to mediate staining; 3) the glioblastoma cells contained HPA52, as measured by enzymatic assay, while melanoma cells that were not stained did not contain HPA52 activity; 4) dexamethasone reduced both the enzymatically determined HPA52 content and the immunofluorescence in parallel, while progesterone affected none of these parameters; 5) we have previously found that culture fluid conditioned by the glioblastoma cells apart from HPA52 does not contain detectable amounts of any protein that binds to anti-HPA52. Several advantages of immunohistochemical detection of plasminogen activators compared with enzyme histochemical methods are discussed, among these that the immunohistochemical method distinguishes between plasminogen activators of different types.
Carbonic anhydrase localization by light and electron microscopy: a comparison of methods.
Authors: Anderson R E , Gay C V , Schraer H
Published: April 2011

The histochemical localization of carbonic anhydrase by Hansson's cobalt-salt method was compared with immunocytochemical localization in the proventriculus (glandular stomach), the chorioallantoic membrane, and in articular and growth-plate cartilages from the domestic hen. Numerous differences were observed. Staining was positive by Hansson's method and negative by immunocytochemistry for the submucosal glands of the proventriculus, articular cartilage cells, resting and proliferating cells of the growth plate, nuclei, and intercellular spaces. Red blood cells stained faintly and inconsistently by Hansson's method. Both methods were in agreement for the cytoplasm of the surface mucosal cells of the proventriculus, the cytoplasm of the villus cavity cells in the chorioallantoic membrane, and in hypertrophic cells of growth-plate cartilage. Acetazolamide usually inhibited the histochemical reaction, even in those sites that, according to other methods, did not contain enzyme. Consequently, acetazolamide inhibition appears to be an unreliable control for the histochemical reaction.
Immunohistochemical evidence for cell surface and Golgi localization of galactosyltransferase in human stomach, jejunum, liver and pancreas.
Authors: Pestalozzi D M , Hess M , Berger E G
Published: April 2011

Immunohistochemical localization of galactosyltransferase (UDP-galactose: 2-acetamido-2-deoxy beta-D-glucopyranose beta (1-4) transferase) in human tissue specimens of gastric and jejunal mucosa, exocrine pancreas, and liver was carried out at the light microscopic level using affinity purified rabbit anti-human milk galactosyltransferase antibodies. Intracellular localization of galactosyltransferase in epithelial cells appeared as a triangular compact structure close to the apical pole of the nucleus. In hepatocytes, the enzyme was found in discrete spots in the cytoplasm between the nuclei and the bile canaliculi. In addition to the intracellular, juxtanuclear location an intense reaction at the luminal part of the cell surface was found in the lining epithelium of the stomach, in enterocytes of the jejunal villus tips, and in ductular cells of the pancreas. Enterocytes located in the middle portion along the cryptvillus gradient exhibited cytoplasmic staining adjacent to the brush borders. Basolateral membranes appeared negative. Little or no enzyme could be demonstrated in cells belonging to the connective tissue. These results show that secretory cells contain a Golgi apparatus which can be visualized at the light microscopic level by virtue of its content in galactosyltransferase. Presence of galactosyltransferase antigen on the surface of certain cells supports the assumption that ectoglycosyltransferases do exist, at sites, however, apparently not involved in cell contact and adhesion.
Direct immunocytochemistry with a horseradish peroxidase-conjugated monoclonal antibody against substance P.
Authors: Boorsma D M , Cuello A C , van Leeuwen F W
Published: April 2011

The procedure for the isolation and conjugation of the anti-substance P monoclonal antibody NC1/34 with the enzyme horseradish peroxidase (HRP) is described. This resulted in a molecular complex of monoclonal antibody/HRP of 1:1. This conjugate was of approximately 400,000 daltons, as estimated by gel chromatography. Practically all the isolated antibody was coupled to HRP. The conjugate was tested both in a model system where CNBr-activated Sepharose beads were coupled to substance P and on fixed tissue preparations from the rat spinal cord and medulla oblongata. The conjugate revealed staining in nerve fibers in areas known to contain substance P. The best immunohistochemical results were obtained by prolonged incubations at 12 degrees C in the presence of 0.1% Triton X-100. The preabsorption of the conjugate with substance P obliterated the reaction.
Visualization of anti-sperm plasma membrane IgG and Fab as a method for localization of boar sperm membrane antigens.
Authors: Russell L D , Peterson R N , Russell T A
Published: April 2011

A simple method for ultrastructural localization of sperm surface antigens by direct visualization of bound antibodies is presented. Anti-sperm plasma membrane (ASPM) immunoglobulin (Ig) G, visualized in tissues treated with an osmium:ferrocyanide mixture, projected 11-13 nm from the surface and ASPM Fab fragments projected 8-10 nm from the surface. The density of IgG labeling, as subjectively estimated, corresponded to indirect immune fluorescein isothiocyanate, indirect immunoferritin, and sperm-vesicle labeling patterns. Agglutination of sperm vesicles and sperm were demonstrated and the linking antibody visualized. A second antibody on protein A directed against ASPM IgG made the immunologic tag more apparent and indicated, in disrupted sperm preparations, labeling of both sides of the plasma membrane. The method provides for easy and sensitive localization of sperm surface antigens at the ultrastructural level and is presently being used to localize specific sperm antigens.
Electron microscopic visualization of cathepsin D using mercury-labeled pepstatin as an enzyme inhibitor.
Authors: Yamato S , Hirabayashi Y , Sugihara H , Uematsu H
Published: April 2011

Pepstatin, a specific inhibitor of pepsin, cathepsin D (E), renin, etc., was used in a new method to demonstrate the sites of enzymes. Pepstatin (Pst) was covalently attached to glutathione (GSH), using a dicyclohexylcarbodiimide, and CH3Hg+ was then put in the residue of SH for electron microscopic observation. Pst-GS-HgCH3 thus obtained was nonisotopical, had a very low molecular weight (about 1,200 daltons), and still almost completely retained its inhibitory activity. Sections of rat liver (less than 1 mm3 thick), prefixed by glutaraldehyde, were incubated in the prepared reaction medium. Cathepsin D, located in the lysosomes, was demonstrated by this compound, but when the sections were preincubated with pepstatin, this was not the case. These results demonstrate that mercury-labeled pepstatin is a useful reagent for the histochemical staining of acid proteases for electron microscope.
Renal gamma-glutamyltranspeptidase: In situ antiluminal localization.
Authors: Dass P D , Misra R P , Welbourne T C
Published: April 2011

Renal localization of gamma-glutamyltranspeptidase was studied in functioning kidneys perfused with the gamma-glutamyl substrates, gamma-glutamyl-p-nitroanilide and N(gamma-L-glutamyl) 2-naphthylamide. In kidneys perfused with gamma-glutamyl-p-nitroanilide, 90% of the breakdown products appear in the blood (perfusate) rather than urine. The addition of the acceptor substrate glycylglycine to the perfusate stimulated gamma-glutamyl-p-nitroanilide breakdown 3-fold, both on the blood and urine sides, while the appearance of gamma-glutamylglycylglycine confirmed that the activity responsible stems indeed from gamma-glutamyltranspeptidase. Ureter ligation reduced the glomerular filtration rate to less than 10% of the control, while the breakdown of gamma-glutamyl-p-nitroanilide decreased only 5%. Kidneys perfused with N(gamma-L-glutamyl) 2-naphthylamide and the diazonium salt demonstrated staining of the glomeruli; isolated glomeruli and microvessels gave heavy staining when incubated with the histochemical substrates. These results demonstrate the association of a significant portion of the total renal gamma-glutamyltranspeptidase activity with the microvascular compartment.
Some fluorescent counterstains for neuroanatomical studies.
Authors: Schmued L C , Swanson L W , Sawchenko P E
Published: April 2011

Methods for counterstaining neural tissue that contains fluorescent markers have been developed. Acridine orange is useful for localizing cells that are retrogradely labelled with the fluorescent tracers true blue, bisbenzimide, and nuclear yellow because at low concentrations it yields a green Nissl stain when excited with blue, but not with ultraviolet, light; since the tracers fluoresce only when exposed to ultraviolet light, they are not masked by the counterstain. In addition, counterstaining at pH 2 increases bisbenzimide fluorescence considerably. Ethidium bromide is useful for immunohistochemistry (IHC) because it yields a bright red Nissl counterstain when excited by green light, and is only faintly visible when the fluorescein marker is excited with blue light, or when ultraviolet excitation is used. Ethidium bromide is therefore a good counterstain for fluorescent retrograde tracer and for combined IHC-retrograde tracer studies as well. Certain dyes are also useful for studies of the normal morphology of neural tissue. For example, bisbenzimide and nuclear yellow at low concentrations produce a brilliant Nissl stain at pH 2, and stain only nuclei at pH 7.2. The latter procedure may be particularly useful for cell counts. Finally, neutral red, astrazone red, and safranin-O differentially stain cells amd myelinated fibers, producing fluorescence analogs of the Klüver-Barrera stain.
Evaluation of immunoperoxidase-stained tissue sections with an electrophoresis densitometer.
Authors: Witorsch R J
Published: April 2011

The application of the Phoroscope electrophoresis densitometer for the objective evaluation of immunoperoxidase-stained tissue sections at the light microscope level is described. The instrument directs a beam of light through the specimen (Kodachrome photomicrograph slides) and projects an absorbance pattern (as peaks and valleys) on an oscilloscope screen. The height of a peak is proportional to the optical density of a stained area, while the width of the peak is proportional to the diameter of an area. The utility of the densitometer was tested in sections of rat pituitary gland immunostained with NIAMDD antiserum against rat prolactin to reveal prolactin cells and sections of rat ventral prostate pretreated with NIAMDD rat prolactin and then immunostained to reveal Golgi region-localized prolactin binding sites in epithelial cells. The densitometer quantitated (by relative absorbance) the changes in immunostaining in serial sections of rat pituitary gland reacted with different dilutions of prolactin antiserum and in serial sections of rat ventral prostate pretreated with different concentrations of rat prolactin. In addition, the densitometric pattern provided information regarding the frequency and pattern of immunostaining within a particular microscopic field. Potential uses of an electrophoresis densitometer for the quantitation and standardization of immunohistochemistry are discussed.
Immunocytochemical identification of dye-injected cells.
Authors: Michaels R L
Published: April 2011

Lucifer Yellow CH may be injected into pancreatic islet cells and visualized in Epon sections of the embedded tissue both before and after plastic removal and immunocyto-chemical staining. The dye retains its fluorescence, clearly marking the injected cell and adjacent dye-coupled cells, but does not interfere with the indirect immunofluorescent staining patterns that are characteristic of the islet cells
Immunocytochemical localization of mitochondrial proteins in the rat hepatocyte.
Authors: Bendayan M , Shore G C
Published: April 2011

Two mitochondrial proteins, carbamyl phosphate synthetase (CPS) and a structural membrane protein, OMM-35, were specifically localized in the hepatocyte mitochondrial matrix and inner and outer membranes, respectively, using the protein A-gold technique. Three embedding media, Epon, glycol methacrylate (GMA), and Lowicryl K4M, were tested for their ability to provide good ultrastructural preservation of mitochondrial membranes, while at the same time retaining protein antigenicity in embedded liver. Epon embedding proved to be relatively unsuitable, since mitochondrial membranes were poorly preserved. GMA and Lowicryl however gave excellent ultrastructural preservation and retained protein antigenicity sufficiently well to enable the localization of the structural membrane protein. Both qualitative and quantitative immunocytochemical demonstration of CPS have ascertained its localization to the rat hepatocyte mitochondrial matrix. The enzyme was undetectable, however, in the mitochondria of liver endothelial cells. OMM-35 was specifically located in the mitochondrial membranes and the quantitative evaluation confirms the biochemical data that OMM-35 is clearly enriched in the outer mitochondrial membrane. OMM-35 was detected in mitochondria of both hepatocytes and endothelial cells. The labeling of a relatively minor structural membrane protein such as OMM-35 gives and indication of the high sensitivity of the protein A-gold immunocytochemical technique.
Fluorescence demonstration of dipeptidyl peptidase I (cathepsin C) in skeletal, cardiac, and vascular smooth muscles.
Authors: Stauber W T , Ong S H
Published: April 2011

Dipeptidyl peptidase I (cathepsin C) was demonstrated histochemically in soleus, extensor digitorum longus, and cardiac muscles but not in vascular smooth muscle cells of the caudal artery of the rat. The enzyme using Pro-Arg-4-methoxy-beta-naphthylamide as the substrate was found in discrete granules in the striated muscles. The activity was greatest in the soleus muscle, with less activity observed in cardiac tissue, and only a few reactive sites observed in the extensor digitorum longus muscle. Under identical conditions no activity was observed associated with vascular smooth muscle cells. Dipeptidyl peptidase I activity was inhibited completely by 1mM HgCl2 in the incubation solutions and not preserved following conventional chemical fixation techniques.
Immunohistological localization of desmin, the muscle-type 100 A filament protein, in rat astrocytes and Müller glia.
Authors: Dahl D , Bignami A
Published: April 2011

The distribution of glial fibrillary acidic (GFA) protein and desmin was compared in cryostat sections of rat brain, spinal cord, and eye by immunofluorescence and peroxidase-antiperoxidase (PAP) staining. Desmin antisera were raised to antigen purified from chicken gizzard. In rat brain and spinal cord, GFA protein and desmin were selectively localized in astrocytes. Neurons and axons were not stained. The only difference between GFA and desmin antisera was the staining of smooth muscle in small arteries with anti-desmin. It was only in retinal glia that a difference in the localization of the two proteins was apparent. As previously reported, only the glia limitans on the inner surface of the retina was demonstrated with GFA antisera in the normal eye. With anti-desmin Müller fibers spanning the whole thickness of the retina were stained. It is concluded that GFA and desmin form two distinct systems of 100 A filaments in astroglia, as previously reported for GFA and vimentin.
Ultrastructural localization of salivary acidic proline-rich proteins from Macaca fascicularis.
Authors: Kousvelari E E , Oppenheim F G , Cutler L S
Published: April 2011

Using an indirect immunoferritin method, the subcellular distribution of acidic proline-rich proteins (MPRP) in the acinar cells of the macaque parotid and submandibular glands was defined. MPRP was found in the "spherule" substructure of the secretory granules as well as in specific Golgi transfer vesicles and in vesicles budding from the Golgi apparatus. The data suggest that the acidic proline-rich proteins of the primate Macaca fascicularis are synthesized and packaged by conventional exocrine mechanisms. In addition, these proteins appear to be packaged and transferred to the secretory granules of parotid and submandibular gland acinar cells as discrete aggregates that remain as a separate "spherule" area in the secretory granules.
Fibronectin involvement in granulation tissue and wound healing in rabbits.
Authors: Repesh L A , Fitzgerald T J , Furcht L T
Published: April 2011

This study describes the distribution of fibronectin and its association with reticulin fibers (type III collagen) and hyaluronic acid in shallow rabbit wounds. Linear incisions were made dorsally with a surgical blade. Animals were sacrificed and 1,2,3,4,5, and 8 day wounds were examined using peroxidase-antiperoxidase to localize affinity-purified antibodies to fibronectin. Tissue samples were also stained with hematoxylin and eosin in addition to silver stains for reticulin, and Alcian blue for hyaluronic acid. After wounding, the incision filled with a fibrin clot that stained positively for fibronectin. The underlying dermis and adjacent, unwounded dermis also contained fibronectin. Epidermal cells that migrate from the wound margin between the clot and the dermis were in direct association with fibronectin in these wound components. By 72 hr, epidermal continuity was reestablished. Early granulation tissue formation was apparent just below the epidermis 5 day wounds. Fibronectin was observed in the matrix surrounding individual fibroblasts and codistributed with reticulin fibers and hyaluronic acid in both 5 and 8 day wounds. Granulation tissue of 8 day wounds stained intensely for fibronectin and extended to a greater depth in the reticular dermis. Dense fibrillar networks of fibronectin and fibroblasts were aligned parallel to the epidermis, giving the granulation tissue a highly structured and organized appearance. Fibroblasts contained fibronectin and were surrounded by less fibronectin at the wound periphery than within the granulation tissue. These findings suggest that fibronectin may be important in the reconstruction of tissues during repair by functioning as an extracellular scaffold for migrating cells.
Immunohistochemical localization of acid alpha-glucosidase in rat liver.
Authors: Iwamasa T , Ninomiya N , Hamada T , Fukuda S
Published: April 2011

Acid alpha-glucosidase (E.C. 3.2.1.3) was purified more than 60,000-fold from rat liver. Antibody was obtained by injection of this pure enzyme into rabbits with Freund's complete adjuvant. The resultant anti-acid alpha-glucosidase immunoglobulin (Ig) G was digested with pepsin and then F(ab')2 was treated with 2-mercaptoethanol. Coupling of Fab' to horseradish peroxidase was performed according to the method of Wilson and Nakane. Light microscopic observation of the immunohistochemical localization of this enzyme in rat hepatocytes revealed small granular deposits of diaminobenzidine reaction products. The reaction diffusely observed in the hepatocyte cytoplasm of any area. Under the electron microscope, the reaction precipitates were found to be located on the lysosome membrane, particularly on the inner side of the membrane, as small dots. The small vesicles were strongly positive for this reaction. Occasionally positive reaction were also demonstrated in the lumen of the secondary lysosomes. However, the Golgi and its associated structures did not show a positive reaction.
A histochemical and X-ray microanalysis study of calcium changes in insect flight muscle degeneration in Solenopsis, the queen fire ant.
Authors: Jones R G , Davis W L , Vinson S B
Published: April 2011

Potassium pyroantimonate histochemistry, coupled with ethyleneglycoltetraacetic acid (EGTA)-chelation and X-ray microprobe analysis, was employed to localize intracellular calcium binding sites in the normal and degenerating flight musculature in queens of Solenopsis, the fire ant. In normal animals, calcium distribution was light to moderate within myofibrils and mitochondria. In the early contracture stages of the insemination-induced degeneration, both myofilament and mitochondrial calcium loading was markedly increased. In the terminal stages of myofibril breakdown, only Z-lines (isolated or in clusters) with an associated filamentous residue persisted. These complexes were also intensely calcium positive. This study further documents the presence of increased sarcoplasmic calcium during muscle necrosis. Surface membrane defects, mitochondrial calcium overload, and calcium-activated proteases may all be involved in this "normal" breakdown process.
Plasmalemma-bound nucleoside diphosphatase as a cytochemical marker of central nervous system (CNS) mesodermal cells.
Authors: Vorbrodt A W , Wisniewski H M
Published: April 2011

Nucleoside diphosphatase (NDPase) activity, using inosine-5'-diphospbate (IDP) as a substrate, was studied by light and electron microscope cytochemistry in brain and spinal cord of normal mouse. After cytochemical incubation for the enzyme, neurons, astroglial and oligodendroglial cells contained reaction product in the cisternae of endoplasmic reticulum (ER) and/or in the trans elements of the Golgi apparatus. In contrast, reaction product for NDPase was concentrated on the outer surface of the plasmalemma of microglial cells, endothelial cells of microvessels, pericytes, and smooth muscle cells of the arteriole wall. An occasional microglial cell contained sparse reaction product in the flattened saccules of the Golgi apparatus. The pattern of enzyme distribution for these cell types was the same for all parts of the nervous system examined in this study. These data suggest that in cells of ectodermal origin, NDPase is intracellular in location, whereas in cells of mesodermal origin, this enzyme is bound to the plasmalemma. The usefulness of this cytochemical reaction for NDPase as a marker of microglial and other cells in brain pathology is discussed.
Immunohistochemical localization of renin in the human kidney.
Authors: Faraggiana T , Gresik E , Tanaka T , Inagami T , Lupo A
Published: April 2011

By using an antiserum to purified human renal renin, renin was localized immunocytochemically in the human kidney under normal and various pathological conditions by the unlabeled antibody enzyme light microscope: (LM) and protein A-gold colloid electron microscope (EM) procedures. In the normal kidney, renin was confined to the epithelioid cells of the afferent arteriole of the juxtaglomerular apparatus (JGA). These cells were small and few, and always in the immediate neighborhood of the glomerulus. Fine structural analysis showed renin only in the secretion granules of the epitheloid cells. All granules within a given cell were stained with comparable intensity. In cases of renal artery stenosis (ischemic kidney) and of Bartter's syndrome, renin-positive epithelioid cells were larger, showed increased staining intensity, and were often found along the afferent arteriole at some distance from the glomerulus. Again, by electron microscopic observation, renin was seen only in secretion granules of epithelioid cells. In all of the above pathologic cases, plasma renin activity was very high. However, in the other nephropathies studied, renin staining in the kidney resembled that seen in normal kidneys, even when levels of plasma renin activity were quite high.
Demonstration of lactoperoxidase in paraffin-embedded lactating bovine breast by immunocytochemical techniques.
Authors: Bussolati G , Gugliotta P , Papotti M
Published: April 2011

Immunocytochemical methods have been applied to the demonstration of lactoperoxidase (LP) in the lactating bovine breast. Trypsin digestion and a MAGIC (Wachsmuth ED: Histochemie, 37:251, 1973) procedure, using anti-LP serum and then LP solution in sequence, made the sensitive and specific demonstration of LP in formalin-fixed paraffin-embedded sections possible. Peroxidase of the breast has been shown to be inducible by estrogen stimulation and it is considered to be a possible marker of estrogen dependency in breast carcinomas. The present technique, applied to human tissues, even on fixed and embedded biopsies, might make possible the detection of LP in human breast cancers and, thereby, an evaluation of estrogen dependency of such cancers.
Electron microscopic immunoperoxidase staining of insulin using 4-chloro-1-naphthol after osmium fixation.
Authors: Baskin D G , Mar H , Gorray K C , Fujimoto W Y
Published: April 2011

Ultrastructural localization of insulin in B cells of guinea pig pancreas was accomplished after osmium fixation with an immunoperoxidase procedure that utilized 4-chloro-1-naphthol (CN) in the substrate solution. The principal features of this protocol were: a) osmium tetroxide postfixation; b) omission of hydrogen peroxide "etching"; c) use of CN instead of diaminobenzidine in the substrate solution; d) elimination of osmium tetroxide after the substrate reaction; e) uranyl acetate and lead citrate counterstaining. This procedure produces intense specific staining with low background using highly dilute antiserum, and appears to be useful for postembedding immunoperoxidase staining of a variety of peptide antigens in osmium-fixed tissue.
Improved mass determination of isolated biological objects by transmission electron microscopy and scanning microdensitometry.
Authors: Linders P W , Zahniser D J , Stols A L , Stadhouders A M
Published: April 2011

A modification of the classical method of Bahr and Zeitler for dry mass determination of isolated biological objects is described. The method uses a conventional transmission electron microscope (TEM) and a computer-controlled scanning microdensitometer. The performance of the modified method has been investigated by applying it to model specimens (polystyrene latex spheres). The method offers significant improvements over the existing TEM-based mass determination method.
Patterns of filipin-sterol complex distribution in intact erythrocytes and intramembrane particle-aggregated ghost membranes.
Authors: Brown D , Montesano R , Orci L
Published: April 2011

When fixed erythrocytes are exposed to the cholesterol probe, filipin, and freeze-fractured, their membrane is labeled with only a limited number of typical, 25 nm diameter filipin-sterol complexes. In addition, the membrane contains many less distinct filipin-induced perturbations that give it an overall rippled appearance. In contrast, red cell ghosts subjected to conditions that result in an aggregation of their intramembrane particles have large particle-free membrane domains which are completely filled with 25 nm filipin-sterol complexes. These results suggest that some constraint exists in the intact erythrocyte membrane which restricts the formation of typical filipin-sterol complexes.
Silver staining of the nucleolus organizer regions (NOR) requires clusters of sulfhydryl groups.
Authors: De Capoa A , Ferraro M , Lavia P , Pelliccia F , Finazzi-Agrò A
Published: April 2011

Silver stainability of nucleolus organizer regions (NORs) appears to be correlated with the presence of grouped sulfhydryl (SH) side chains of proteins. In fact, heavy metals with high affinity for SH groups, such as Hg and Cu, do prevent the silver staining reaction. Ferricyanide, which is known to oxidize SH to disulfides, also prevents any further reaction with silver. On the other hand, alkali and reducing agents (mercaptoethanol, cyanide) do not affect silver stainability of the NORs. These results show that the silver staining reaction is not related to disulfide or persulfide groups and that alkali-soluble, acidic nuclear proteins per se do not play a major role in this process.
Glial fibrillary acidic (GFA) protein in Schwann cells: fact or artifact?
Authors: Dahl D , Chi N H , Miles L E , Nguyen B T , Bignami A
Published: April 2011

Antisera to the glial fibrillary acidic (GFA) protein stained a subpopulation of Schwann cells in cryostat sections of rat sciatic nerve by indirect immunofluorescence and by the peroxidase-antiperoxidase (PAP) procedure. The staining pattern was entirely different from that obtained with vimentin antisera, which uniformly decorated endoneurial tubes. Electron microscopic examination of sciatic nerve provided a possible explanation for the relatively small number of Schwann cells decorated by GFA antisera: 10 nm filaments were mainly confined to Schwann cell processes surrounding nonmyelinated axons. A marked increase in GFA-positive Schwann cells and in Schwann cells containing filaments by electron microscopy was observed in sciatic nerves undergoing Wallerian degeneration. Conversely, immunochemical procedures failed to demonstrate the presence of antigen reacting with GFA antisera in extracts of sciatic nerve, both normal and degenerated. These include absorption experiments, double immunodiffusion, immunoaffinity chromatography, and immunoradiometric assay. Two explanations may be considered for these findings: i) Schwann cell intermediate filaments and GFA protein share common antigenic determinants, the immunohistological methods being more sensitive to detect cross-reactivity as compared to immunochemical procedures on tissue extracts; and ii) the binding of anti-GFA to Schwann cell 10 nm filaments is not due to immunological cross-reactivity.
Light and electron microscopic immunocytochemical localization of clathrin in rat cerebellum and kidney.
Authors: Lin C T , Garbern J , Wu J Y
Published: April 2011

The precise cellular and subcellular locations of coated vesicle protein, clathrin, in rat kidney and cerebellum have been visualized by immunocytochemical techniques. In the renal tubular epithelia, clathrin-positive products were found on both free ribosomes and on those attached to rough endoplasmic reticulum (RER) and the nuclear envelope. No clathrin was observed in the cisternae of RER or the Golgi apparatus. Clathrin-positive reaction products could also be seen on coated pits, coated vesicles, Golgi-associated vesicles, basolateral cell membrane, the ground substance, and in the autophagic vacuoles. In cerebellar Purkinje and granule cell bodies, reaction products were seen localized on coated vesicles, on the budding areas from the Golgi-associated membrane and Golgi-associated vesicles. Furthermore, the membrane of the multivesicular body, the bound-ribosomes, and the ground substance were also stained. In the myelinated axon, the clathrin appeared to be concentrated on certain segments and seemed to fill in the space between neurotubules and some vesicles. In certain synaptic terminals clathrin was often seen attached to presynaptic vesicles, presynaptic membrane, and post-synaptic membrane. However, in most mossy fibers, some synaptic vesicles were not stained. These observations suggest that clathrin is synthesized on bound and free ribosomes and discharged into the cytosol where it becomes associated with a variety of ground substances and assembles on coated pits, coated vesicles, Golgi-associated vesicles, presynaptic vesicles, and pre- and postsynaptic membranes. Clathrin may be finally degraded in autophagic vacuoles.
Use of lectins for detection of glycoconjugates in the glandular cells of the human bronchial mucosa.
Authors: Mazzuca M , Lhermitte M , Lafitte J J , Roussel P
Published: April 2011

Paraffin-embedded human bronchial biopsies, obtained in macroscopically healthy areas, were examined using nine peroxidase-bound lectins. These were either isolated or purified by affinity column chromatography (Ulex europeus, Triticum vulgare, Glycine max, and Arachis hypogatea lectins) or commercial preparations (Lotus tetragonolobus, Canavalia ensiformis, Helix pomatia, Phaseolus vulgaris, and Lens culinaris lectins) and conjugated to peroxidase (except for concanavalin A). These labeled lectins were used as specific molecular probes to localize differences in carbohydrate-containing components present in the different types of glandular cells of human bronchial mucosa. The choice of fixative was crucial and tests used for this study have shown that Carnoy's solution seems to be the most appropriate solution to preserve mucous glycoproteins in situ. Comparison of the affinity of several lectins for human bronchial glycoproteins and for bronchial mucosa demonstrates the predominance of serum-type glycoproteins in serous cells of the submucosal glands and mucin-type glycoproteins in mucous cells of the submucosal glands and in goblet cells of the bronchial epithelium. Furthermore the data obtained with some lectins, such as Helix pomatia agglutinin, suggest that there are some differences in the mucins synthesized by goblet cells and by mucous cells.
Ultrastructural localization of antigenic sites on osmium-fixed tissues applying the protein A-gold technique.
Authors: Bendayan M , Zollinger M
Published: April 2011

The protein A-gold immunocytochemical technique has been modified to allow labeling of cellular antigenic sites on osmium-fixed or postfixed tissues. Several strong oxidizing agents have been found able to restore protein antigenicity on osmicated tissue thin sections. According to the fine structural preservation and intensities of labeling, pretreatment with sodium metaperiodate gave optimal results. Pancreatic secretory proteins (and/or proproteins) as well as insulin (and/or proinsulin) were localized over perfectly preserved rough endoplasmic reticulum (rER), Golgi apparatus, and secretory granules of the corresponding pancreatic cells; carbamyl phosphate synthetase and catalase were revealed over liver mitochondria and peroxisomes, respectively. In addition to the higher resolution in the labeling obtained using osmium-fixed tissues, the present modification confers an additional advantage to the protein A-gold technique by allowing labeling on tissues processed for routine electron microscopy.
Acrolein: a fixative for immunocytochemical localization of peptides in the central nervous system.
Authors: King J C , Lechan R M , Kugel G , Anthony E L
Published: April 2011

Acrolein was examined as an alternative fixative to formaldehyde for immunocytochemical localization of neuropeptides in the rat brain. A brief (5 min) vascular perfusion with a 5% acrolein solution allowed the identification of thyrotropin-releasing hormone (TRH), vasoactive intestinal peptide (VIP), somatostatin (SRIF), neurotensin (NT), methionine enkephalin (Menk), adrenocorticotropic hormone (ACTH), tyrosine hydroxylase (TH), and luteinizing hormone-releasing hormone (LHRH) in fibers and perikarya within the central nervous system of the rat using the peroxidase-antiperoxidase (PAP) technique. Acrolein appears to be particularly valuable for immunocytochemistry, as it 1) stabilizes heterogeneous peptides and proteins rapidly and effectively, 2) retains antigenicity, and 3) preserves morphological detail.
Immunocytochemical localization of fibronectin in human fibroblast cultures using a cell surface replica technique.
Authors: Irish P S , Hasty D L
Published: April 2011

The pericellular fibronectin-containing matrices of human foreskin fibroblasts cultured in ascorbate-supplemented medium were examined using surface replicas. An extensive filamentous network is present over and between adjacent cells, with a considerable amount at points of cell-to-cell contact. Indirect immunocytochemical localization of the distribution of fibronectin and procollagen type III within the matrix was done using the peroxidase-antiperoxidase (PAP) sandwich technique. The PAP molecule with the surrounding diaminobenzidine reaction product appears as a globular particle of approximately 39 nm in surface replicas. The apparent size of the marker was larger (60-80 nm) when bound to pericellular fibronectin, due presumably to the binding of more than one PAP complex to each fibronectin molecule. The immunocytochemical data suggest that fibronectin is a component of most, if not all, matrix fibrils. Some of the smallest filaments of the matrix (5-10 nm) exhibit a periodic, beaded appearance, with a repeat distance of approximately 70-100 nm. After either anti-fibronectin or anti-procollagen type III labeling, the filaments were decorated at regular 70-100 nm intervals with the globular marker. We suggest that the periodicity may be due to fibronectin molecules bound to collagen microfibrils at regular intervals. Our results demonstrate the usefulness of combined surface replica and immunocytochemical techniques for analysis of matrix components of cultured cells.
Mannose-specific binding sites for horseradish peroxidase in various cells of the rat.
Authors: Straus W
Published: April 2011

Mannose-specific binding sites for horseradish peroxidase (HRP) were studied in fixed sections of various tissues by a method reported previously. Liver sinusoidal cells, mast cells of lymph nodes, and alveolar macrophages of the lung and skin fibroblasts were main cell types showing mannose-specific binding of HRP. Macrophages, fibroblasts, and mast cells in the connective tissue of other organs also showed the reaction. However, macrophages of the spleen, and cultured 3T3 cells and L-cells did not give the reaction. The specificities of the binding reaction were studied by determining the approximate concentrations of competing sugars that suppressed the specific binding of HRP. It was found that the endogenous lectins in macrophages, fibroblasts, mast cells, and liver sinusoidal cells showed similar specificities toward various carbohydrates. D-Mannose and L-fucose had the highest affinity toward the lectins (competing ability for the binding of HRP). D-Mannose-6-phosphate, N-acetyl-D-glucosamine, D-glucose, D-ribose, and D-arabinose showed intermediate affinity, whereas D-xylose and D-galactose showed low affinity. Polymerized mannose in mannan and glycoproteins rich in mannose groups (invertase and ribonuclease B) showed much higher affinity to the binding sites than free mannose.
A comparison of eight different chromogen protocols for the demonstration of immunoreactive neurofilaments or glial filaments in rat cerebellum using the peroxidase-antiperoxidase method and monoclonal antibodies.
Authors: Trojanowski J Q , Obrocka M A , Lee V M
Published: April 2011

Eight different previously described chromogen protocols were evaluated with respect to their sensitivity for the visualization of horseradish peroxidase (HRP) in a peroxidase-antiperoxidase (PAP) complex used with the unlabeled antibody method for immunohistochemistry. The protocols were evaluated in a test system that involved the demonstration of immunoreactive neurofilaments (NF) or glial filaments (GF) in paraffin-embedded sections of rat cerebellum using anti-NF or anti-GF monoclonal antibodies (MA). The chromogens included: amino-ethylcarbazole (AEC), diaminobenzidine (DAB), O-tolidine, paraphenylenediamine-pyrocatechol (PPD-PC), and tetramethylbenzidine (TMB). The incubation medium using DAB as the chromogen was employed at neutral pH, at pH 5.1, or at neutral pH with the addition of either cobalt chloride or imidazole to intensify the reaction product. The relative sensitivity of the chromogen protocols was quantitated by comparing the dilution of the anti-NF or anti-GF MA at which NF or GF immunoreactivity was extinguished using each protocol. The results obtained with both the anti-NF and anti-GF MA indicated that DAB with imidazole was the most sensitive chromogen protocol.
Characterization of mononuclear cells in cytocentrifuge and imprint preparations using monoclonal antibodies and an avidin--biotin immunoperoxidase staining system.
Authors: Giorno R
Published: April 2011

The application of an avidin--biotin immunoperoxidase staining system was investigated for the characterization of mononuclear cells in cytocentrifuge and imprint preparations with monoclonal antibodies. The system was found to be very sensitive, permitting the use of high dilutions of monoclonal antibody. The system also permitted the study of single cell morphology. Its utilization should be complementary to the study of patterns of cellular proliferation in frozen section material. Furthermore, it may lend itself to automation for quantitative analysis of cell populations.
Immunohistochemical demonstration of poly(adenosine diphosphate-ribose) synthetase in bovine tissues.
Authors: Ikai K , Ueda K
Published: April 2011

The inter- and intracellular localization of poly(adenosine diphosphate-ribose)(poly(ADP-ribose] synthetase was investigated using an indirect immunofluorescence technique and a specific antibody against the enzyme purified from calf thymus. In various bovine tissues, including liver, heart, pancrease, thyroid, spleen, adrenal, and skeletal muscle, the specific immunofluorescence of poly(ADP-ribose) synthetase was localized exclusively in the nucleus. Immunostaining was inhibited by preabsorption of the antibody with purified calf thymus poly(ADP-ribose) synthetase. Nuclear immunofluorescence appeared to be more prominent in the marginal area than in the central region in most nuclei. This staining pattern is similar to that of naturally occurring poly(ADP-ribose). In bovine peripheral blood the immunofluorescence of poly(ADP-ribose) synthetase was detected in nuclei of lymphocytes, but not in granulocytes, in agreement with the finding that the enzymatic activity of poly(ADP-ribose) synthetase was barely detectable in nuclei isolated from granulocytes.
Distribution and synthesis of type V collagen in the rat mammary gland.
Authors: Warburton M J , Monaghan P , Ferns S A , Hughes C M , Rudland P S
Published: April 2011

In the 100-day-old virgin and lactating rat mammary glands, type V collagen is mainly present in the interstitial connective tissue and in association with blood vessels. It is not present in the basement membrane region surrounding the ducts in mature virgin glands but is present in this region in neonatal and lactating glands. Ultrastructural localization of type V collagen reveals that it is mainly located on the basal surface (i.e., the surface in contact with the basement membrane) of epithelial but not myoepithelial cells. In addition, type V collagen is located on some interstitial collagen fibers and on a large number of granules that are in close proximity to the basal surface of both epithelial and myoepithelial cells. Immunofluorescence and biochemical studies indicate that several clonal mammary fibroblastic cell lines synthesize type V collagen in vitro. In some cell lines, type V collagen is secreted as an extensive fibrillar meshwork on the surface of the cells, whereas in other cell lines, it is secreted beneath the cells around their periphery. A number of mammary epithelial and myoepithelial-like cells, however, do not synthesize type V collagen in vitro.
A method for examining the endothelial cytoskeleton in situ using immunofluorescence.
Authors: Rogers K A , Kalnins V I
Published: April 2011

A simple technique is described for producing en face preparations of endothelial cells (EC) from large blood vessels fixed in situ that are suitable for studying the distribution of specific antigens in EC by immunofluorescence. This method has permitted us to examine the distribution of various components of the cytoskeleton, including microtubules (MT), centrioles, and microfilaments (MF) in EC in vivo.
Method for analysis of cellular DNA content of paraffin-embedded pathological material using flow cytometry.
Authors: Hedley D W , Friedlander M L , Taylor I W , Rugg C A , Musgrove E A
Published: April 2011

A method has been developed that allows flow cytometry to be used for measuring the cellular DNA content of paraffin-embedded human tumors. Thick (i.e., 30 micron) sections were cut from tissue blocks using a microtome and dewaxed in xylene. The sections were then rehydrated by sequentially immersing them in 100, 95, 70, and 50% ethanol before finally washing in distilled water. Single cell suspensions were then prepared by incubation in 0.5% pepsin, pH 1.5, at 37 degrees C for 30 min. The cells were counted, washed, and stained with 1 microgram/ml 4',6'-diamidino-2-phenylindole for 30 min, and DNA content was measured using an ICP 22 flow cytometer. There was a good correlation between the DNA histograms produced using this method and those obtained using unfixed tissue from the same tumor stained with ethidium bromide plus mithramycin. This method allows the retrospective study of archival material where the clinical outcome is already known, and it should, therefore, be particularly useful for determining the prognostic significance of abnormal DNA content measured by flow cytometry.
Actin in peripheral rat lung: S1 labeling and structural changes induced by cytochalasin.
Authors: Tsilibary E C , Williams M C
Published: April 2011

Actin was identified with S1-labeling in type I and II cells, pericytes, and capillary endothelial cells in the peripheral lung of the adult rat. In type II cells abundant actin was present in the apex of cells, in microvilli, and in close association with lamellar bodies near the cell surface. Lamellar bodies secreting their content into the lumen of alveoli were always surrounded by a thick layer of actin-like material. In specimens treated with cytochalasin D the surface of type I, type II and contractile interstitial cells became irregular. In type II cells, lamellar bodies were no longer surrounded by actin-like material and no exocytic profiles of lamellar bodies were encountered. In type II cells actin filaments may be involved in moving lamellar bodies through the cytoplasm and/or in their secretion into alveoli. These observations also suggest that intact actin filaments may be required for maintenance of cell shape in various lung cells and that cells containing actin may be capable of limited contraction.
Chromaffin reactions and fluorometric determination of catecholamines in the neonatal adrenal medulla of the rat.
Authors: Piezzi R S , Bianchi R A , Miranda J C
Published: April 2011

Adrenaline and noradrenaline fluorometric determinations were simultaneously made in adrenal glands of 2-to-10 day-old rat with the histochemical localization of total catecholamines and noradrenaline. In 10-day-old-rats the increase is three times higher for adrenaline and four times higher for noradrenaline compared to 2nd day of age. Chromaffin cells of 2-to-10-day-old rats showed a low granular population preferentially located in the periphery of the cells. Seven days after birth the chromaffin granules were numerous and dispersed in extense areas of the cytoplasm. The dichromate reactions for total catecholamines were positive from the 5th day of age when the fluorometric determination was 250 ng of catecholamines (CA) per gland. The glutaraldehyde silver technique for noradrenaline was positive from the 7th day when the fluorometric determination was 35 ng of noradrenaline (NA) per gland. However, at the electron microscope level, this last reaction shows the same silver precipitates on the chromaffin granules in adrenal glands obtained from 4-to-6-day-old rats. These results indicate that the appearance and evolution of the chromaffin positive reaction during post natal development is a direct expression of molecular changes during maturation of the granular complex.