bluuebunny/crossref_metadata_2025 huggingface dataset:
Immunocytochemical localization of glycoprotein hormones in the rat anterior pituitary. A light and electron microscope study using antisera against rat bet subunits: a comparison between preembedding and postembeeding methods.
Authors: Tougard C , Picart R , Tixier-Vidal A
Published: April 2011

The binding sites of antisera (anti) to the beta (beta) subunits of rat follicle-stimulating hormone (rFSH), rat luteinizing hormone (rLH), and rat thyroid-stimulating hormone (rTSH) have been localized in rat anterior pituitaries by immunocytochemistry using light and electron microscopy. With the light microscope, LHbeta and FSHbeta were found in the same cells, which were violet after the alcian blue-periodic acid Schiff (AB-PAS) staining. TSHbeta was found in polygonal or stellate cells that were blue after AB-PAS. With the electron microscope, the thyrotropic cells contained very small secretory granules. LHbeta and FSHbeta were found in various types of cells (types A and B and their intermediate forms), which had previously been identified as gonadotropic cells. On serial ultrathin sections using the postembedding method the same cells and even some granules inside these cells were stained by both anti-rLHbeta and anti-rFSHbeta. A comparison of binding sites of anti-rLHbeta was performed using the preembeeding and the postembeeding methods. Antigenicity was observed on secretory granules whatever the method used. However, binding sites of anti-rLHbeta were detected inside the cisternae of the rough endoplasmic reticulum only with the preembedding method.
Microfluorimetric scanning of sympathetic nerve fibers: quantification of neuronal and extraneuronal fluorescence with the aid of histogram analysis.
Authors: Schipper J , Tilders F J , Wassink R G , Boleij H F , Ploem J S
Published: April 2011

A high resolution (0.5 micrometer), two-dimensional microfluorimetric scanning procedure was used to quantitate the formaldehyde-induced fluorescence of noradrenaline in the iris of the rat. Scanning of large areas (50 x 50 micrometer) in the sympathetic innervated dilator muscle revealed an overlap between measurements of nerve fibers and of background (smooth muscle). In order to discriminate between both populations, the scan data were converted into fluorescence histograms (256 fluorescence classes, and subjected to mathematical analysis. The characteristics of the background histogram were obtained from scans of iris preparations devoid of fluorescent nerve fibers (pretreatment of the donor rat with reserpine or sympathetic denervation). A curve-fitting program was applied on those fluorescence classes in the normal iris histogram that represent measurements on pure background and resulted in a full background histogram. After subtraction of this background histogram from the original histogram, a nerve fiber histogram was obtained. The validity of the algorithm was evaluated by scanning iris preparations with varying background intensities. The results showed that quantification of the nerve fiber fluorescence was independent of variation in the background.
Biophysical cytochemical investigations of intracellular heparin in neoplastic mast cells.
Authors: Nakamura N , Hurst R E , West S S , Menter J M , Golden J F , Corliss D A , Jones D D
Published: April 2011

The thermodynamic binding parameters for intracellular heparin-acridine orange (AO) complexes were determined for Furth murine mastocytoma cells and were found to agree with 1) results from binding studies on heparin-AO complexes in solution, and 2) with biochemical analyses of the cells. The cells exhibited cooperative binding with a binding constant of 1.18 x 10(6) M-1. The cooperative binding constant of heparin-AO in 1 mM buffer was found to be 1.13 x 10(6) M-1. The addition of 1 mM NaCl to heparin-AO system in vitro detectably decreased the cooperative binding constant. Low ionic strength is the only condition in solution under which the cell and solution binding constants are equal. The cells have an average of 1.2 x 10(-14) mol of AO binding sites per cell. Using the biochemically measured heparin content per cell and the amount of AO bound by heparin in solution, 8 x 10(15) mol of sites/cell can be attributed to heparin. The remaining cellular binding sites (4 x 10(-15) mol of sites per cell) are essentially all accounted for by AO binding to DNA, the amount of which is calculated from its previously determined thermodynamic binding parameters. A theoretical isotherm, calculated from the binding parameters of both heparin-AO in solution and DNA-AO complexes in situ, agreed closely with the isotherm experimentally determined for the Furth mastocytoma cells. Ligand-binding analysis yields a binding constant, which may aid in identification of cellular bipolymers, and the number of ligand binding sites per cell. The latter is a measure of the amount of a given intracellular biopolymer present.
The distribution of lysosomal cathepsin D in cardiac myocytes.
Authors: Decker R S , Decker M L , Poole A R
Published: April 2011

Lysosomal cathepsin D has been localized with the electron microscope employing an indirect immunohistochemical method using peroxidase labeled, monospecific antibody Fab' subunits. The acid proteinase has been demonstrated within secondary lysosomes of cardiac myocytes and interstitial cells, but not in components of the Golgi apparatus or endoplasmic reticulum. Incubations with a variety of peroxidatic inhibitors suggests that the staining that is observed in secondary lysosomes is attributable to the peroxidase-labeled antibody and not to endogenous oxidation of DAB. The protocol outlined here provides a reproducible method to localize the major lysosomal acid proteinase of the heart at the subcellular level.
Immunofluorescence and histochemical localization of glucosephosphate isomerase in neural tissues.
Authors: Oster-Granite M L , Gearhart J
Published: April 2011

The distribution of glucosephosphate isomerase (GPI, D-glucose-6-phosphate ketol isomerase) in mouse nervous tissue has been determined at the light microscopic level by immunofluorescence and histochemical procedures. The fluorescence procedure, which utilizes anti-GPI antibodies, detected lower levels of GPI than the histochemical procedure, which relies upon the catalytic activity of the enzyme. The distribution of GPI in nervous tissue is very similar to that of hexokinase. High levels of GPI were found in the Purkinje cells, the molecular layer, and the glomeruli of the granular layer in the cerebellar cortex; the pontine nuclei and the inferior olivary nuclei of the pons and medulla; the neurons of the thalamus and hypothalamus; the pyramidal cells, the dentate nuclei, and Ammons' horn of the cerebral cortex; the ventral horn cells of the spinal cord; and ventricular cells, choroid plexus cells, and the leptomeninges. The neuropil throughout the central nervous system (CNS) stained uniformly with moderately high levels of GPI. No GPI was observed in the myelin sheaths of the CNS.
Back to back autoradiography: a method for ultrastructural analysis of light microscopic autoradiographs.
Authors: Kaplan N B , Smith L J , Brody J S
Published: April 2011

A method for studying radiolabeled cells is described that combines the simplicity of light microscopic autoradiography with the high resolution of electron microscopy. Serial thin (600 A) and thick (1 micron) sections are placed on Formvar-coated slot grids and glass slides, respectively. Labeled cells are visualized on the thick section by autoradiography and may then be studied in the electron microscope by locating the corresponding fields on the grid. This technique permits accurate ultrastructural identification and analysis of radiolabeled cells, yet avoids the need for electron microscopic autoradiography.
Demonstration of species difference of placental alkaline phosphatase isozymes in acetone-fixed paraffin-embedded tissues.
Authors: Dempo K , Kottel R H , Fishman W H
Published: April 2011

The best preservation and discrimination of alkaline phosphatase isozymes by means of amino acid inhibition and heat treatment was obtained in sections of acetone-fixed paraffin-embedded tissues as compared to tissues fixed either in 95% ethanol or standard formalin. The results are illustrated in a study differentiating the isozymes of human and mouse placenta.
Ultrastructural localization of basic proteins in cytoplasmic granules of rat eosinophils and mast cells.
Authors: Pimenta P F , Loures M A , de Souza W
Published: April 2011

The postformalin ammoniacal silver (AS) and the ethanolic phosphotungstic acid (EPTA) methods were used to localize basic protein at the ultrastructural level in cytoplasmic granules of rat eosinophils and mast cells isolated using a Metrizamide gradient. Intense reaction was seen in the granules of EPTA-treated eosinophils. Following incubation of the cells for 2 hr in EPTA alone, the matrix was stained. After longer incubation (10 hr), however, both the matrix and core were stained. Cytoplasmic granules of the mast cell show a slight or negative reaction with EPTA. With the AS technique, a large number of silver particles were seen in the nucleus of both eosinophils and mast cells. The mast cell cytoplasmic granules showed intense reaction, while those from eosinophils showed no clear reaction. Acetylation of the cells under conditions sufficient to block most free amino groups prio to EPTA or AS treatment greatly reduced (EPTA) or abolished (AS) the reaction. The results indicate 1) that eosinophil granules contain basic proteins both in the matrix and the core, 2) that the mast cell granules contain a basic protein (probably the alpha-chymotrypsin-like enzyme), which reacts strongly with AS, and 3) that the AS and EPTA methods have different specificities.
ATPase in mature and differentiating phloem and xylem.
Authors: Cronshaw J
Published: April 2011

A cytochemical study using a lead precipitation technique has been made of the distribution of adenosine triphosphatase (ATPase) in mature and differentiating phloem and xylem cells of Nicotiana tabacum and Pisum sativum. The sites of ATPase localization in tobacco phloem were the plasma membrane, endoplasmic reticulum, mitochondria, dictyosomes, plasmodesmata, and the dispersed P proteins of mature sieve elements. In pea phloem sieve elements ATPase was localized in the endoplasmic reticulum, but was not associated with the P proteins or plasma membranes at any stage of their differentiation. In pea transfer cells ATPase activity was associated with the endoplasmic reticulum at all stages of their differentiation and with the plasma membrane of transfer cells that had formed wall ingrowths. In xylem cells of both tobacco and pea the patterns of ATPase activity was similar. At early stages of differentiation ATPase activity was associated with the plasma membrane and the endoplasmic reticulum. At intermediate stages of differentiation ATPase activity continued to be associated with the endoplasmic reticulum, but was no longer associated with the plasma membrane. At later stages of xylem element differentiation ATPase activity was associated with disintegrating organelles and with the hydrolyzing cell walls.
Immunoelectron microscopy localization of immunoglobulin G in human placenta.
Authors: Lin C T
Published: April 2011

Immunoelectron microscopy of IgG molecules in human mature placenta has shown that IgG bound to microvillar surfaces and the inner wall of endocytotic vesicles of syncytiotrophoblasts. The endocytotic vesicles, containing both bound and unbound IgG molecules, tended to fuse with each other or with other cellular organelles, particularly with lysosomes. The phagolysosomes were more abundant in the basal regions of the cells. Apparently some IgG molecules were not digested by lysosomal enzymes. Vesicles with residual IgG were found to fuse with the basal and basolateral cell membrane and to discharge their contents into the extracellular space by exocytosis. It is suggested that IgG molecules were transported through the trophoblastic basement membrane and the interstitial space by diffusion to the endothelial basement membrane. The IgG molecules then migrated into the fetal vascular lumen via endothelial gaps and interendothelial spaces.
Are there indications for a granule-free form of vasopressin?
Authors: Krisch B
Published: April 2011

Following a short immobilization the supraoptic perikarya exhibit peroxidase-antiperoxidase (PAP)-labeled rough endoplasmic reticulum and the intercellular clefts in the basal nuclear area are distinctly labeled by PAP molecules. The fibers in this basal part of the supraoptic nucleus as well as those of the internal zone of the median eminence, beside few polymorphic, positively strained granules, contain a labeled tubular network or free PAP on fuzzy material.
A squash technique demonstrating embryonic nuclear cleavage of the nematode Caenorhabditis elegans.
Authors: Gossett L A , Hecht R M
Published: April 2011

A simple squash technique was developed which permits the observation of individual nuclei during embryogenesis of Caenorhabditis elegans. The technique consists of placing several two-cell stage embryos on a subbed slide in a droplet of M-9 salt buffer and incubating them in a sealed humidity chamber at 16.4 degrees C for increasing time intervals. The embryos are then squashed, fixed, and stained with Hoechst 33258. Rate of cleavage at 25.0 degrees C is 1.8 times faster than that at 16.4 degrees C. This yields superimposable growth curves upon correction for temperature. An initial lag in the rate of nuclear cleavage is followed by a burst of cell proliferation, which continues and then slows before 550-580 cells are produced at 4 to 5 hr at 25 degrees C. The squash size increases with cell number and reaches a maximum at about the 400-cell stage when early morphogenesis begins. The second half of embryogenesis is characterized by histogenesis in which the cells are held more tightly together, individual nuclei become less distinct, and the squash size decreases to a minimum as a small worm is formed.
Immunohistochemical study of the blood-brain barrier. Production of an artifact.
Authors: Sparrow J R
Published: April 2011

The permeability to normal serum proteins of blood vessels in the central nervous system (CNS) was studied by means of the peroxidase-antiperoxidase immunohistochemical technique. Frozen sections were cut in a cryostat and then fixed briefly in 95% ethanol. The localization of serum proteins outside blood vessels in the brain was shown to be an artifact produced during the preparation of cryostat sections. It was concluded that such cryostat sections are unsuitable for studies of vascular permeability in the CNS.
Dithiocarbamylation in histochemistry: carbon disulfide as a reagent for the visualization of primary amino groups with the light and electron microscope.
Authors: Tandler C J
Published: April 2011

Tissue amino groups undergo dithiocarbamylation on treatment (12 hr, 22-25 degrees C) with a carbon disulfide:triethylamine:tetrahydrofuran (1:1:0.5, v/v) mixture. After washing with tetrahydrofuran:triethylamine (4:1, v/v) followed by tetrahydrofuran:pyridine (4:1,v/v) and ice-cold distilled water tissues were immersed into ice-cold 10% (w/v) lead acetate to yield yellowish lead dithiocarbamates. At 22-25 degrees C the derivatives obtained from primary amines decompose to lead sulfide. After washing with 5% lead acetate in 10% acetic acid followed by three changes of distilled water tissues are dehydrated and embedded in paraffin or Maraglas as usual. In proteins the reaction is given by the epsilon-amino group of lysine and alpha-terminal amino groups, while nucleic acids do not react. Results are shown in tetrahydrofuran-delipidized ethanol or formalin-fixed 1) plant root tips where brown-colored and electron-dense lead sulfide deposits are localized in condensed chromatin and chromosomes attributable to lysine-rich histones and 2) rat skeletal muscle where an interfibrillar component is visualized in a repeating pattern, possibly related to terminal regions of the sarcoplasmic reticulum.
Localization of proteoglycan monomer and link protein in the matrix of bovine articular cartilage: An immunohistochemical study.
Authors: Poole A R , Pidoux I , Reiner A , Tang L H , Choi H , Rosenberg L
Published: April 2011

Using monospecific antisera and immunofluorescence microscopy, proteoglycan monomer (PG), and link proteins were demonstrated throughout the extracellular matrix of bovine articular cartilage. A narrow band of strong pericellular staining was usually observed for both molecules, indicating a pericellular concentration of proteoglycan monomer: this conclusion was supported by dye-binding studies. Whereas PG was evenly distributed throughout the remaining matrix, more link protein was detectable in interterritorial sites in middle and deep zones. Well-defined zones of weaker territorial staining for link protein stained strongest for chondroitin sulfate. Trypsin treatment of cartilage resulted in a loss of most of the PG staining, but some selective retention of link protein, particularly around chondrocytes in the superficial zone at and near the articular surface. This residual staining was largely removed if sections were fixed after chondroitinase treatment. After extraction of cartilage with 4M guanidine hydrochloride, only PG remained and this was concentrated in the superficial zone. These observations are shown to support the concept of aggregation of PG and link protein with hyaluronic acid (HA) in cartilage matrix, and the binding of PG and link protein to HA, which is attached to the chondrocyte surface. Culture of cartilage depleted of PG and link protein by trypsin demonstrated that individual chondrocytes can secrete both PG and link proteins and that the organization of cartilage matrix can be regenerated in part over a period of 4 days.
Flow cytometric analysis of double-stranded RNA content distributions.
Authors: Frankfurt O S
Published: April 2011

A staining procedure is described for quantitative analysis of cellular RNA content distributions by flow cytometry (FCM). Cells were fixed in ethanol, treated with DNAse and stained with propidium iodide. FCM analysis showed that more than 90% of the fluorescence resulted from the double-stranded RNA (dsRNA) bound fluorochrome. The dsRNA content distributions were measured in HeLa S3 cells in culture 24-120 hr after plating, in L1210 ascitic leukemic cells 1-7 days after transplantation, and in regenerating bone marrow 3-7 days after injection of cyclophosphamide. In all three cell populations, maximal dsRNA content was observed during the period of most active cell proliferation, as determined by the S-phase index derived from the DNA histograms. In the dsRNA distributions in L1210 leukemia cell populations 1-2 days after transplantation, two separate peaks were evident, representing weakly fluorescent, presumably nontumor cells, and intensively fluorescent tumor cells. The amount of dsRNA-bound fluorochrome was significantly greater in L1210 cells than in normal and regenerating bone marrow cells and also greater in spontaneous thymic lymphoma cells than in normal thymic cells.
Use of a trypan blue assay to measure the deoxyribonucleic acid content and radioactive labeling of viable cells.
Authors: Allison D C , Ridolpho P
Published: April 2011

A simple method of determining cellular viability, DNA content, and isotopic labeling is described. Cells are sedimented onto slides and fixed in a mixture of trypan blue and paraformaldehyde which stains nonviable cells a dark blue color, whereas viable cells exclude the dye. The staining pattern is stable for at least 6 months and persists after the Feulgen reaction, allowing the selection of viable cells for cytophotometric DNA determinations. We ascertained cellular incorporation of a 3H-thymidine label by photomapping cells prior to cytophotometry and relocating these cells after preparation of autoradiographs. The method gives accurate quantitative DNA and labeling values of mouse thymocytes labeled in vitro and may be of value in studies of tumors containing a large proportion of necrotic cells.
Immunohistochemical demonstration of poly(adenosine diphosphate-ribose) in nuclei of various rat tissues.
Authors: Ikai K , Ueda K , Hayaishi O
Published: April 2011

Natural distribution of poly(adenosine diphosphate-ribose), a novel macromolecule in eukaryotes, was investigated using an indirect immunofluorescence technique. The antibody, produced in a rabbit toward poly(ADP-ribose), was most reactive with polymers having the chain length of about 25 ADP-ribose units and weakly reactive with short oligomers; it was totally inert with monomers. Immunostaining with this antibody revealed the existence of the polymer in various rat tissues. The immunostaining seems to be specific for poly(ADP-ribose), as judged by its disappearance by preabsorption of the antiserum with purified poly(ADP-ribose) or pretreatment of tissue sections with poly(ADP-ribose)-degrading enzymes. Intensification of the fluorescence by preincubation with nicotinamide adenine dinucleotide (NAD), a substrate for poly(ADP-ribose) synthesis, also supported this view. The immunofluorescence of poly(ADP-ribose) was found exclusively in the nucleus of almost all tissues tested, including liver (adult, newborn, regenerating, and hepatoma), brain, heart, intestine, pancreas, kidney, spleen, testis, thyroid gland, and skeletal muscle. Exceptions were blood cells; little fluorescence was detectable in nuclei of peripheral leukocytes. Only after preincubation with NAD, did lymphocytes and monocytes exhibit fluorescence, however, granulocytes never did exhibit fluorescence. The cells appeared to represent the first instance where poly(ADP-ribose) synthesis activity among eukaryotic cells was missing.
Factors affecting the sensitivity and specificity of the cytochemical reaction for the anti-horseradish peroxidase antibody in lymph tissue sections.
Authors: Straus W
Published: April 2011

Factors which increase the sensitivity and specificity of the cytochemical reaction for the antibody to horseradish peroxidase (HRP) in precursors of plasma cells and in lymphocytes were studied in sections of popliteal lymph nodes of rats. The lymph nodes were removed 3-5 days after a secondary injection of HRP into the footpads and were fixed for 5 hr in a 4% cold formaldehyde solution (Straus W: Histochemistry 53:273, 1977). Brief postfixation of the frozen sections with cold acetone improved the retention of the antigen at the sites of the antibody in the precursor cells, and it improved the quality of fixation without appreciably weakening the antigen-binding capacity of the antibody. The cytochemical reaction for the anti-HRP antibody was intensified by staining with diaminobenzidine (DAB) and H2O2 at pH 5-6, or by staining at pH 7.4 in the presence of imidazole. Imidazole partially inhibited endogenous peroxidase activity. Pretreatment with phenylhydrazine prevented nonspecific background adsorption of HRP. Phenylhydrazine had the additional advantage of inhibiting most of the endogenous peroxidase activity (Straus W: J Histochem Cytochem 20:949, 1972). The intensity of the antibody reaction in the proplasma cells developing in the medullary cords varied greatly depending on the stage of maturation from lymphocytes and blast cells. Many lymphocytes in the cortex of the lymph node showed a strong perinuclear antibody reaction when the tissue sections were postfixed with cold acetone, and the peroxidase complexed to the antibody was visualized by staining with DAB and H2O2 at pH 5-6. The antibody reaction also occurred at the surgace of many lymphocytes when the tissue sections, postfixed with cold acetone, were stained with DAB and H2O2 at pH 7.4 in the presence of imidazole. Other lymphocytes showed a strong surface, perinuclear, and cytoplasmic antibody reaction after staining at pH 5-6 as well as after staining at pH 7.4, while yet other lymphocytes remained unstained.
Ultrastructural localization to 10 nm filaments of an insoluble 58K protein in cultured fibroblasts.
Authors: Cabral F , Willingham M C , Gottesman M M
Published: April 2011

Using a simple procedure, we have isolated a Triton X-100 insoluble protein (58,000 mol wt) from cultured Chinese hamster ovary (CHO) cells, a fibroblastic cell line. The isolated protein, judged homogeneous by two-dimensional gel electrophoresis, was used to elicit antibodies in rabbits. The antiserum obtained is specific for the 58K protein as determined by immunoprecipitation from detergent solubilized 35S-labeled CHO cells and by antibody labeling of SDS solubilized Swiss 3T3 cell extracts separated on a 10% polyacrylamide gel. Indirect immunofluorescence localization with this antiserum in fixed permeabilized Swiss 3T3 cells, whose flat morphology makes them superior for localization studies, reveals filaments that radiate from the perinuclear region to the cell periphery. These filaments were identified as 10 nm filaments by electron microscopic localization using the EGS and ferritin bridge procedures. We conclude that the 58K protein is a major constituent of fibroblast 10 nm filaments. Other intracellular structures were not labeled by this technique.
Receptor-mediated endocytosis of alpha 2-macroglobulin in cultured fibroblasts.
Authors: Willingham M C , Maxfield F R , Pastan I
Published: April 2011

Alpha 2-macroglobulin is internalized into cultured fibroblasts by receptor-mediated endocytosis. This ligand binds initially to diffusely distributed receptors on the cell surface which cluster rapidly into bristle-coated pits. Within a few minutes at 37 degrees C, these complexes are internalized into uncoated cytoplasmic vesicles, called receptosomes, which move about in the cell by saltatory motion. These vesicles interact with the Golgi-endoplasmic reticulum-lysosome system in the cell to deliver the ligand to newly formed lysosomes within 30--60 min.
Postnatal developmental changes in submandibular glands of rats and mice.
Authors: Gresik E W
Published: April 2011

The submandibular glands of mice and rats are not fully developed at birth. In early postnatal life, differentiation of acini takes place before that of granular convoluted tubule (GCT) cells. The latter develop from striated duct cells, and first appear in both species around 15 days of age. In mice their full development gets under way by 20 days of age and is rapid in males and slow in females, resulting in a clear sexual dimorphism in adults. In rats, GCT development is more protracted, and accelerates around 40 days of age, with no sexual dimorphism seen at any time. The course of postnatal development of several GCT cell products is correlated with the cytodifferentiation of these cells. Reliable data are available for the development of amylase, proteases (including kallikrein), renin, epidermal growth factor, and nerve growth factor. Preliminary information exists for a glucagon-like substance. Cytodifferentiation of GCT cells is under hormonal control. Androgens alone can not precociously induce GCT cells, but thyroid hormones can do so, acting either alone or synergistically with androgens.
A reliable Feulgen-acriflavine-SO2 staining procedure for quantitative DNA measurements.
Authors: Tanke H J , van Ingen E M
Published: April 2011

Feulgen-acriflavine-SO2 staining was performed on chicken erythrocytes and rat liver cells under different cytochemical conditions. The obtained results were compared to conventional Feulgen-pararosaniline-SO2 staining by means of scanning cytophotometry and microfluorometry. It was found that if acid ethanol rinsing was included in the Feulgen-acriflavine-SO2 procedure, both Feulgen procedures were fully comparable in specificity and quantitative aspects.
Short time bleaching of fluorescein isothiocyanate. A possible parameter for the specific binding of conjugates in immunofluorescence.
Authors: Schauenstein K , Böck G , Wick G
Published: April 2011

The fluorescence kinetics of fluorescein isothiocyanate (FITC)-antibody conjugates during short (0.01 sec) laser excitations were analyzed for possible information about the immunological specificity of binding to antigenic substrates in direct immunofluorescence assays with antigen-coated Sephadex beads. First results of these investigations suggest marked differences in the bleaching characteristics of specifically and nonspecifically bound FITC conjugates: FITC-anti-bovine serum albumin (BSA) and FITC-anti-rabbit immunoglobulin (Ig) were found to fade significantly more slowly when bound to the respective homologous antigen (anti-BSA to BSA, and anti-rabbit Ig to rabbit Ig) as compared to nonspecifically adherent anti-BSA to rabbit Ig, and anti-rabbit Ig to BSA. Possible implications of these data for discrimination of nonspecific staining in immunofluorescence are discussed.
Comparisons of alveolar and peritoneal macrophages: soluble protein, esterase, dipeptidyl aminopeptidase II, and proteinase inhibitor.
Authors: Allen R C , Sannes P L , Spicer S S , Hong C C
Published: April 2011

The content of soluble protein, nonspecific esterase, dipeptidyl aminopeptidase II (DAP II), and proteinase inhibitors was compared for alveolar (AM) and peritoneal (PM) wash cells of rats. The cells present in the wash fluids were 85-90% macrophages in the peritoneal wash and 95% in the alveolar wash. Macromolecular components were resolved from whole cell homogenates by polyacrylamide gel isoelectric focusing (PAGIF) on horizontal gels and were identified cytochemically. Banding patterns clearly indicated a larger number of esterase zones in peritoneal compared to alveolar macrophages and ten previously unrecognized isozymes of DAP II in peritoneal macrophages with only three evident in their alveolar counterparts. (On whole blood smears, these cytochemically demonstratable enzymes were limited to macrophages, although DAP II was seen also in some mast cell granules). A protein band similar to the M6 band of alpha-1-antitrypsin in human serum was seen both in alveolar and peritoneal wash preparations. In addition, nine other major trypsin-binding protein bands were observed in the peritoneal macrophages, including two bands not observed in the alveolar macrophage extracts.
Intracellular localization of fibronectin by immunoelectron microscopy.
Authors: Yamada S S , Yamada K M , Willingham M C
Published: April 2011

We have localized fibronectin, a major extracellular glycoprotein of cultured fibroblasts, in chick embryo fibroblasts at the ultrastructural level using affinity-purified antibodies to fibronectin. The use of a ferritin bridge procedure permitted quantitation of localization in various organelles. These results provide the first intracellular ultrastructural localization of fibronectin. Extracellular labeling was confined to aggregates and fibrils, with little or no labeling of the plasma membrane. The principal sites of intracellular localization were the rough endoplasmic reticulum and the Golgi apparatus. Treatment of cells with the protein synthesis inhibitors cycloheximide and pactamycin reduced fibronectin localization in the endoplasmic reticulum to 50% of normal levels. Removal of cycloheximide permitted recovery of labeling to 85% of control levels in the endoplasmic reticulum. Similar, but much reduced, changes also occurred in the Golgi apparatus.
A histochemical model dealing with an immobilized glucose oxidase-peroxidase system. The influence of diffusion limitations on histochemical results.
Authors: Malpiece Y , Sharan M , Barbotin J N , Personne P , Thomas D
Published: April 2011

A histochemical model dealing with an immobilized bienzyme system (glucose oxidase-peroxidase) is presented. The model is an artificial proteic membrane obtained by a previously described co-cross-linking process. The kinetic properties of free and immobilized horseradish peroxidase were studied when 3,3'-diaminobenzidine is used as a hydrogen donor substrate. A new direct method was developed for immobilized enzyme activity measurements. Computer simulation based on experimental kinetic parameters was performed in order to discuss electron microscopy results. By changing diffusion limitations, various profiles of insoluble product were visualized inside the proteic film and no geometrical similarity was seen between enzyme distributions and insoluble osmiophilic product patterns.
Elimination of excess smooth endoplasmic reticulum after phenobarbital administration.
Authors: Feldman D , Swarm R L , Becker J
Published: April 2011

Livers from Charles River rats during and after treatment with phenobarbital were studied in order to investigate possible mechanisms involved in the elimination of excess smooth endoplasmic reticulum. The most pronounced structural change during compound administration was proliferation of smooth endoplasmic reticulum; depletion of glycogen and an increase in lipid deposits were also observed. After termination of treatment, these changes were reversed. The appearance of an increased number of autophage vacuoles and lysosomes plus the localization of acid phosphatase reaction product in these bodies suggests autophagy as one possible mechanism for the elimination of excess smooth endoplasmic reticulum. Cytoplasmic blebs and fragments replete with smooth endoplasmic reticulum were observed within the sinusoids. The presence of Kupffer cell cytoplasmic extensions surrounding these fragments and acid phosphatase reaction product within Kupffer cell inclusions suggests heterophagy as another process participating in the removal of excess smooth endoplasmic reticulum.
Fluorescence of yeast vitally stained with ethidium bromide and propidium iodide.
Authors: Corliss D A , White W E
Published: April 2011

Both ethidium bromide and propidium iodide stain growing yeast. As visualized in the fluorescence microscope, ethidium stains the nucleus and cytoplasm in wild type yeast and in those grown in 10% dextrose, with brightly fluorescent cytoplasmic granules being present in both. Under the latter conditions, the mitochondria are repressed but not absent. In rho 0 cells, in which the mitochondrial DNA is absent, ethidium appears to bind to the cell wall or membrane preferentially with no cytoplasmic granules being visible. In all cell types, propidium appears to bind the cell wall or membrane with no cytoplasmic granules being visible in any cell. The staining patterns thus suggest greater differences in the binding of these two types to mitochondrial DNA in situ than is suggested by their in vitro behavior. These differences in binding could explain their different mutagenic capacities..
Immunocytochemical localization of receptor-bound human chorionic gonadotropin in pseudopregnant rat ovaries.
Authors: Kinnunen R , Karjalainen M , Rajaniemi H
Published: April 2011

Localization of receptor-bound human chorionic gonadotropin (hCG) in pseudopregnant rat ovarian cells was studied by the peroxidase-antiperoxidase (PAP) complex method. The animals were injected, 2,6,12, and 24 hr prior to killing with a single intravenous dose of hCG. The hormone localized to the periphery of the luteal and interstitial glandular cells. The luteal cells seemed to bind different amounts of the hormone, which suggests that the number of receptor varies from one cell to another or that the receptors are not similarly accessible to the circulating hormone in all cells. The hormone seemed to disappear unequally from the luteal cells, possibly due to the variable amounts of primarily bound hormone or to different removal mechanisms. The hormone was distributed unevenly at the luteal cell periphery. This observation supports previous findings that hCG preferentially binds to the cell surface regions directed towards the capillary areas. Compatible results were obtained with anti-hCG serum and with antisera against the hCG subunits. Our results support the previous autoradiographic evidence of the plasma membrane localization of luteinizing hormone receptor in the luteal cells, and are compatible with the concept that only a small fraction of the receptor-hormone complexes may be internalized.
Antigenic separation of a nonkinin-generating TAMe esterase from human urinary kallikrein and immunohistochemical comparison of their localization in the kidney.
Authors: Pinkus G S , Ole-Moiyoi O , Austen K F , Spragg J
Published: April 2011

Two urinary enzymes that cleave alpha-N-p-tosyl-L-arginine methyl ester (TAMe) have been separated and utilized to elicit monospecific antisera; only one, urinary kallikrein (urokallikrein), had kinin-generating activity. The nonkinin-generating TAMe esterase and urokallikrein were antigenically unrelated. Immunoperoxidase studies of normal human kidney revealed localization of nonkinin-generating TAMe esterase to epithelial cells of the distal tubule, including the ascending thick limb, the macula densa region, and some areas of convoluted tubule. Immunoreactivity for urokallikrein was confined to reabsorption droplets of proximal tubules and to focal segments of the distal convoluted tubules. Electrophoretic, antigenic, and immunohistochemical studies have established that urokallikrein and a nonkinin-generating TAMe esterase represent two distinct renal distal tubule enzymes.
Enzyme histochemistry of developing rat oral mucosa.
Authors: Sjögren S , Hammarström L , Larsson A
Published: April 2011

The oral mucosa of developing and mature rats was analyzed histochemically for regional enzyme differences. The following enzymes were studied: nonspecific alkaline phosphatase (alkpase), acid phosphatase (acidpase), 5'-nucleotidase (AMPase), adenosine triphosphatase (ATPase), succinate dehydrogenase (SDH), lactate dehydrogenase (LDH), and glucose-6-phosphate dehydrogenase (G-6-pDH). All enzymes were active in the oral mucosa, but regional as well as tissue variations were observed. Epithelium in all regions showed acidpase staining. Oxidoreductases were found in all regions with variations within the epithelium. The epithelium of specific regions stained for alkpase and AMPase, while adjacent epithelium did not. We suggest that the alkpase and AMPase activities are associated with specific functions of the epithelium in these regions.
Increased accuracy of absorption cytophotometric DNA values by control of stain intensity.
Authors: Allison D C , Ridolpho P F , Rasch E M , Rasch R W , Johnson T S
Published: April 2011

A method of improving absorption cytophotometric cellular DNA values by making measurements on Feulgenstained cells at optimal stain absorbances has been developed. Stain intensity can be controlled either by alteration of the Feulgen staining reaction or by selection of "off-peak" wavelengths of light for cytometry. The use of chicken red blood cells as an internal standard, and of a computerized cytometer for the measurements, allows selection of the appropriate off-peak light wavelengths, correction for staining variability at different sites on the same slide, and rapid calculation of cellular DNA values. Cytometry can also be performed at controlled absorbance levels on autoradiographs of 3H-thymidine-labeled cells to allow direct study of the DNA content of nonlabeled G1/G0 and G2/M cells. Use of this technique on mixtures of mouse thymocytes, spleen cells, bone marrow cells, and liver cells gave essentially identical values for G1/G0 cellular DNA content, with coefficients of variation of less than 3%.
Sciatin: immunocytochemical localization of a myotrophic protein in chicken neural tissues.
Authors: Oh T H , Sofia C A , Kim Y C , Carroll C , Kim H H , Markelonis G J , Reier P J
Published: April 2011

A mytotrophic protein (sciatin) purified from chicken sciatic nerves has "trophic" or "maintenance" effects on cultured muscle. We have elicited a specific antiserum against purified sciatin in rabbits. Using this antiserum, we investigated the distribution of sciatin in embryonic and adult chicken tissues by an unlabeled peroxidase-an-tiperoxidase method at the light microscopic level. The antiserum stained adult chicken neural tissues in situ and cultured embryonic chick neurons. Staining was intense in the cell bodies of spinal cord neurons and the axoplasm of sciatic nerves. These was reaction product seen in the outer margins of myelin sheaths that corresponded to the Schwann cell cytoplasm. Cerebral cortical neurons were weakly stained by the antiserum. No staining was apparent in oligodendrocytes or astrocytes. Nonneural tissues, such as skeletal, smooth and cardiac muscle, kidney, and liver, were also unstained by the antiserum. Cultured spinal cord neurons, cerebral cortical neurons, and sensory neurons were stained immunocytochemically by the antiserum. There was no reaction product seen in the glial cells that are usually present in neuronal cultures or cultured cells from liver, kidney, skeletal muscle, smooth muscle, and cardiac muscle. Our results thus show that the myotrophic protein is localized in neuronal perikarya and their processes in vivo as well as in vitro.
Influences of fixatives on the immunodetection of calcitonin in mouse "C" cells during pre- and post- natal development.
Authors: Treilhou-Lahille F , Cressent M , Taboulet J , Moukhtar M S , Milhaud G
Published: April 2011

The presence of calcitonin in fetal and postnatal mouse "C" cell was demonstrated by radioimmunoassays of fetal and adult thyroid extracts and by immunohistochemistry following the use of Bouin's fixative from 18 days postcoitum onwards. However, after fixation with formaldehyde no calcitonin could be detected in fetal or early postnatal samples, although it was still easily detectable in adult tissues treated with this fixative, raising the possibility that there may be age-dependent differences in the chemistry or physiological state of intracellular calcitonin. Experiments conducted with several other fixatives and the results of the radioimmunoassays suggest that the loss of calcitonin reactivity is not due to differences in the anti-genic sites of the molecule at the two stages in development. Neither can the positive detection of fetal material treated with Bouin's fixative be due to any action of this liquid. However, the inhibiting action of the aldehydes could be related to the preservation of the granule membranes in the fetal "C" cells: our ultrastructural studies show that the granules can be morphologically classified in dense-core and homogeneous matrix. Such granule membranes are abundant in the young and adult "C" cell and would be reactive, while the small dense-core granules are prevalent in the fetus and would be unreactive, confirming the hypothesis of a difference of structure of the granule membrane and not of calcitonin between fetal and late postnatal "C" cells.
Feulgen-deoxyribonucleic acid analysis of rabbit aortic smooth muscle cells using scanning- integrating microdensitometry.
Authors: Anthony A , Hollis T M , Penman J A , Zerweck C , Doebler J A
Published: April 2011

A procedure entailing the use of the Feulgen reaction is described for precise quantification of nuclear DNA levels in smooth muscle cells (SMC) of paraffin-processed microtome sections of the rabbit aorta. It was established that maximal, stable, and reproducible Feulgen-DNA (F-DNA) staining of SMC nuclei is achieved using 3.5 N HCl hydrolysis of 30-50 min prior to staining of aortic sections in Schiff reagent for 60 min at 22 degrees C. Scanning-integrating microdensitometry of Feulgen-stained SMC revealed that the tunica media is comprised of a relatively homogeneous population of cells with between 0.3 and 1% of the SMC nuclei yielding 3C or 4C (tetraploid) F-DNA levels, depending on location within the aortic wall. The nuclear chromatin in inner medial SMC was found to be in a more dispersed state than that of outer SMC (using nuclear area and nuclear susceptibility to acid hydrolysis as indices of chromatin dispersion). A linear correspondence was evidenced between nuclear area and nuclear F-DNA stainability throughout the tunica media. The observation that the lumenal portion of the tunica media contains a greater abundance of SMC with large, vesicular nuclei is interpreted as reflecting a greater metabolic reactivity of this compartment relative to that of SMC bordering the tunica adventitia.
Immunohistochemical localization of retrogradely and anterogradely transported wheat germ agglutinin (WGA) within the central nervous system of the rat: application to immunostaining of a second antigen within the same neuron.
Authors: Lechan R M , Nestler J L , Jacobson S
Published: April 2011

Immunohistochemical localization of retrogradely transported wheat germ agglutinin (WGA) is proposed as a sensitive histochemical technique to identify point to point connections within regions of the central nervous system. Injections of WGA into the median eminence of the hypothalamus and the caudate-putamen complex, respectively, were performed to illustrate that this material is rapidly transported over long distances and accumulates within the cytoplasm of neuronal perikarya and their processes. The applicability of this technique to identification of a second antigen within immunoreactive-WGA-labeled neurons is also demonstrated by sequential immunostaining of tyrosine hydroxylase within dopamine-containing cells of the mesencephalon, ipsilateral to an injection in the caudate-putamen complex. This technique is of use in characterizing bioaminergic neurons in the central nervous system and may also be of use in characterizing peptidergic neurons.
Prolonged incubation time in immunohistochemistry: effects on fluorescence staining of immunoglobulins and epithelial components in ethanol- and formaldehyde-fixed paraffin-embedded tissues.
Authors: Brandtzaeg P
Published: April 2011

By prolonging the incubation time from 30 min to 20 hr at room temperature, fluorochrome conjugates may be applied at about ten times higher dilution and yet produce specific immunofluorescence staining of enhanced intensity. This modification of the direct method is important for reagent economy and, in addition, affords improved staining features for all antigens tested in formaldehyde-fixed tissues. It is particularly valuable when paired staining is used to characterize lymphoproliferative B-cell processes in pathological routine material; a clear-cut distinction between polyclonal and monoclonal expression of cytoplasmic immunoglobulin (Ig) is obtained, and cells giving rise to a false staining pattern are easily pinpointed. It is likewise advantageous to use prolonged incubation with conjugate for the localization of Ig-producing and Ig-bearing B cells in saline-extracted ethanol-fixed tissues, and the same holds true for Ig and C3 in immune-complex deposits. Also IgE on the surface of mast cells in tissues from atopic subjects is visualized distinctly with this modification. However, the localization or epithelial components is not consistently improved in ethanol-fixed tissues when the incubation time is prolonged; secretory products such as lactoferrin, lysozyme, amylase, and secretory component (SC) are not always immobilized sufficiently by ethanol fixation to avoid diffusion artifacts and a substantial loss from the cytoplasm. Differences in intracellular storage probably contribute to the variable antigen stability.
Localization of organ-specific antigens in mouse lung by light and electron microscopy.
Authors: Nathrath W B , Rowlatt C , Detheridge F
Published: April 2011

Rabbit antisera were raised against whole homogenate and a saline soluble fraction of C57BL mouse lung. After absorption with other mouse organs, both antisera gave specific staining of bronchiolar epithelial and single alveolar cells, using the indirect immunofluorescence and indirect immunoperoxidase techniques on both acetic-alcohol-fixed paraffin-embedded and unfixed frozen 5 micrometer sections. Improved resolution by light microscopy was achieved with 1 micrometer sections of formaldehyde-fixed Araldite-embedded material stained by an indirect immunoperoxidase method after the Araldite had been treated with saturated alcoholic NaOH for 5 min. Clara cells and type II pneumocytes were identifiable as sites of the lung-specific immune reaction. At the electron microscopic level the reaction was localized over the granules of bronchiolar Clara cells. The lamellar bodies of type II pneumocytes were extracted in the ultrathin sections and no immune reaction was observed.