scholarly journals Combination of horseradish peroxidase and lucifer yellow staining for selective labeling of neurons at the electron microscopic level.

1991 ◽  
Vol 39 (11) ◽  
pp. 1579-1583 ◽  
Author(s):  
L Nonnotte ◽  
A Buisson ◽  
F Nagy ◽  
M Moulins
Keyword(s):  
Horseradish Peroxidase ◽  
Lucifer Yellow ◽  
Electron Microscopic Level ◽  
Reaction Products ◽  
Stomatogastric Nervous System ◽  
Electron Microscopic ◽  
Double Labeling ◽  
Selective Labeling ◽  
Experimental Neuroanatomy ◽  
Labeling Method

We have developed a new double labeling method for electron microscopy to characterize selectively two physiologically identified neurons on the same preparation. The stomatogastric nervous system of crustaceans was used to test the distinguishing staining characteristics of the two labels. Neurons were labeled on one side with horseradish peroxidase (HRP) and on the other side with Lucifer yellow (LY). After blue light irradiation of the tissue in the presence of diaminobendizine, the two labeled neurons could be easily observed and discriminated on the same section by the two different reaction products. This simple technique of double labeling is useful in experimental neuroanatomy for the detailed study of synaptic relationships.

scholarly journals Adsorption of horseradish peroxidase, ovomucoid and anti-immunoglobulin to colloidal gold for the indirect detection of concanavalin A, wheat germ agglutinin and goat anti-human immunoglobulin G on cell surfaces at the electron microscopic level: a new method, theory and application.

1977 ◽  
Vol 25 (11) ◽  
pp. 1187-1200 ◽  
Author(s):  
W D Geoghegan ◽  
G A Ackerman
Keyword(s):  
Horseradish Peroxidase ◽  
Concanavalin A ◽  
Colloidal Gold ◽  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Indirect Detection ◽  
Electron Microscopic Level ◽  
Specific Cell ◽  
Major Protein ◽  
Electron Microscopic

A method is described for the adsorption of selected macromolecules to colloidal gold which is then used as an electron dense marker for the indirect detection of specific cell surface molecules. Membrane bound concanavalin A, which binds specific sugars on horseradish peroxidase, and wheat germ agglutinin, which binds specific sugars on ovomucoid are detected indirectly with gold labeled horseradish peroxidase and ovomucoid, respectively. Goat anti-human IgM on blood lymphocytes is detected with gold labeled rabbit anti-goat IgG. In the preparation of colloidal gold labeled proteins, the problems of flocculation of colloidal gold by proteins and nonadsorption of proteins to colloidal gold, are solved through a combination of concentration of protein and pH variable adsorption isotherms, which allows one to determine the conditions for adsorption of proteins to colloidal gold. Adsorption is pH dependent, the pH conditions correlating with the isoelectric point(s) of the major protein fraction(s); adsorption is influenced by interfacial tension, solubility and by the electrical charge on the molecules. Colloidal gold is inexpensive and preparation of a useful label is rapid, reproducible and the results easily quantitated from electron micrographs.

X-Ray Analysis of Cytochemical Reaction Products in Synaptic Areas

1976 ◽  
Vol 34 ◽  
pp. 6-7 ◽  
Author(s):  
J. Wood
Keyword(s):  
Electron Microscopic Level ◽  
Junctional Complex ◽  
Reaction Products ◽  
Electron Microscopic ◽  
Cytochemical Localization ◽  
X Ray ◽  
The Past ◽  
Electron Dense Deposit ◽  
Dense Deposit ◽  
Cytochemical Reaction

Specific cytochemical reactions have been instrumental in the illucidation of compounds within tissues, whether these compounds are hormones, enzymes, or molecules, such as certain nerve transmitter agents. Many cytochemical reaction products depend upon some complex, which is an electron dense deposit. Several types of cytochemical procedures can be used to visualize agents related to synaptic transmission at the junctional complex. One method which has been used with considerable success has been the cytochemical localization of biogenic amines (BAs), i.e., norepinephrine (NE) and dopamine (DA). For the past few years, a chrome complex formed with certain BAs and glutaraldehyde has been utilized to localize BAs at the electron microscopic level and the specificity of the reaction has been verified biochemically.

scholarly journals Benzidine dihydrochloride as a chromogen for single- and double-label light and electron microscopic immunocytochemical studies.

1986 ◽  
Vol 34 (8) ◽  
pp. 1047-1056 ◽  
Author(s):  
S Lakos ◽  
A I Basbaum
Keyword(s):  
Horseradish Peroxidase ◽  
Retrograde Tracing ◽  
Primary Antibody ◽  
Electron Microscopic ◽  
Double Labeling ◽  
Bluish Green ◽  
Double Label ◽  
Immunocytochemical Studies ◽  
Anterograde And Retrograde Tracing

Although very sensitive chromogens have been adapted for localization of horseradish peroxidase in anterograde and retrograde tracing studies, they have not been successfully applied in immunocytochemical studies. This report describes a protocol which uses benzidine dihydrochloride (BDHC) as the chromogen for light (LM) and electron microscopic (EM) immunocytochemical studies. The protocol is comparable to that used for tetramethylbenzidine, except that the pH of the reaction is above 6.0. At the LM level, the BDHC reaction product is bluish-green and crystalline. Both the color and form of the product are readily distinguished from the reddish-brown DAB reaction product. LM double-labeling studies are therefore feasible. The use of BDHC also increases significantly the sensitivity of the immunoreaction. Higher fixative concentrations can be used, less detergent is necessary, and higher primary antibody dilutions are possible. By osmicating at 45 degrees C in an s-collidine buffer it is possible to preserve the soluble BDHC reaction product for EM analysis. Immunoreactive cells are particularly well labeled with this new protocol. The BDHC crystals are easily detected at the EM level and can be distinguished from flocculent DAB reaction product. This feature makes EM double-labeling studies possible.

scholarly journals Inter- and intracellular relationship of substance P-containing neurons with serotonin and GABA in the dorsal raphe nucleus: combination of autoradiographic and immunocytochemical techniques.

1986 ◽  
Vol 34 (6) ◽  
pp. 735-742 ◽  
Author(s):  
R Mâgoul ◽  
B Onteniente ◽  
A Oblin ◽  
A Calas
Keyword(s):  
Substance P ◽  
Dorsal Raphe Nucleus ◽  
Dorsal Raphe ◽  
Raphe Nucleus ◽  
Electron Microscopic Level ◽  
Gamma Aminobutyric Acid ◽  
Electron Microscopic ◽  
Double Labeling ◽  
Relationship Of ◽  
Dorsal Raphé

Double-labeling experiments were performed at the electron microscopic level in the dorsal raphe nucleus of rat, in order to study the inter- and intracellular relationship of substance P with gamma-aminobutyric acid (GABA) and serotonin. Autoradiography for either [3H]serotonin or [3H]GABA was coupled, on the same tissue section, with peroxidase-antiperoxidase immunocytochemistry for substance P in colchicine-treated animals. Intercellular relationships were represented by synaptic contacts made by [3H]serotonin-labeled terminals on substance P-containing somata and dendrites, and by substance P-containing terminals on [3H]GABA-labeled cells. Intracellular relationships were suggested by the occurrence of the peptide within [3H]serotonin-containing and [3H]GABA-containing cell bodies and fibers. Doubly labeled varicosities of the two kinds were also observed in the supraependymal plexus adjacent to the dorsal raphe nucleus. The results demonstrated that, in addition to reciprocal synaptic interactions made by substance P with serotonin and GABA, the dorsal raphe nucleus is the site of intracellular relationships between the peptide and either the amine or the amino acid.

scholarly journals Membrane-cytoskeleton dynamics in rat parietal cells: mobilization of actin and spectrin upon stimulation of gastric acid secretion.

1989 ◽  
Vol 108 (2) ◽  
pp. 441-453 ◽  
Author(s):  
F Mercier ◽  
H Reggio ◽  
G Devilliers ◽  
D Bataille ◽  
P Mangeat
Keyword(s):  
Gastric Acid ◽  
Dynamic Equilibrium ◽  
Gastric Gland ◽  
Cytoskeletal Proteins ◽  
Parietal Cells ◽  
Electron Microscopic Level ◽  
Apical Surface ◽  
Electron Microscopic ◽  
Double Labeling ◽  
Acid Secreting

The gastric parietal (oxyntic) cell is presented as a model for studying the dynamic assembly of the skeletal infrastructure of cell membranes. A monoclonal antibody directed to a 95-kD antigen of acid-secreting membranes of rat parietal cells was characterized as a tracer of the membrane movement occurring under physiological stimuli. The membrane rearrangement was followed by immunocytochemistry both at the light and electron microscopic level on semithin and thin frozen sections from resting and stimulated rat gastric mucosa. Double labeling experiments demonstrated that a specific and massive mobilization of actin, and to a lesser extent of spectrin (fodrin), was involved in this process. In the resting state, actin and spectrin were mostly localized beneath the membranes of all cells of the gastric gland, whereas the bulk of acid-secreting membranes appeared diffusely distributed in the cytoplasmic space of parietal cells without any apparent connection with cytoskeletal proteins. In stimulated cells, both acid-secreting material and actin (or spectrin) extensively colocalized at the secretory apical surface of parietal cells, reflecting that acid-secreting membranes were now exposed at the lumen of the secretory canaliculus and that this insertion was stabilized by cortical proteins. The data are compatible with a model depicting the membrane movement occurring in parietal cells as an apically oriented insertion of activated secretory membranes from an intracellular storage pool. The observed redistribution of actin and spectrin argues for a direct control by gastric acid secretagogues of the dynamic equilibrium existing between nonassembled (or preassembled) and assembled forms of cytoskeletal proteins.

A combined pre- and post-embedding double labeling in the central nervous system with good tissue preservation

1991 ◽  
Vol 49 ◽  
pp. 276-277
Author(s):  
A.M. Milroy ◽  
D.D. Ralston
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Room Temperature ◽  
Osmium Tetroxide ◽  
Microscopic Level ◽  
Electron Microscopic Level ◽  
Electron Microscopic ◽  
Double Labeling ◽  
Highly Sensitive ◽  
The Central Nervous System

Multiple labeling at the electron microscopic level is routinely done in various parts of the central nervous system. We demonstrate that the pre-embedding tetramethylbenzidine (TMB) reaction for visualizing horseradish peroxidase (HRP) of Olucha and the slow osmication of Henry combined with a post-embedding nonetching immunogold method will also preserve good ultrastructure. Furthermore, the post-embedding immunocytochemistry of some neurotransmitters, i.e. gammaaminobutyric acid (GABA), can be done months after the tissue has been reacted for HRP and embedded in regular epon.Pre-embedding histochemistry:The use of TMB as a chromagen for the demonstration of neuronally transported HRP has both the advantage of being highly sensitive and of producing very specific needle-like crystals. Olucha et al demonstrated that one could further stabilize this reaction product with amonium heptamolybdate. Unfortunately the next step, fixation with regular osmium tetroxide, often resulted in the loss of the reaction product. However, the slow osmication with a lower pH (5.5) in the phosphate buffer at room temperature as recommended by Henry et al prevented this loss, and at the same time resulted in well preserved ultrastructure.

scholarly journals Four unlabeled antibody bridge techniques: a comparison.

1981 ◽  
Vol 29 (12) ◽  
pp. 1397-1404 ◽  
Author(s):  
P Ordronneau ◽  
P B Lindström ◽  
P Petrusz
Keyword(s):  
Horseradish Peroxidase ◽  
Gamma Globulin ◽  
Staining Intensity ◽  
Reaction Products ◽  
Electron Microscopic ◽  
Microscopic Studies ◽  
Immunocytochemical Techniques ◽  
Unlabeled Antibody ◽  
Shape And Size

Four unlabeled antibody immunocytochemical techniques, the "single bridge" (Avrameas S: Immunocytochemistry 6:825, 1969; Mason TE, Phifer RF, Spicer SS, Swallow RS, Dreskin RD: J Histochem Cytochem 17:190, 1969a; Sternberger LA, Cuculis JJ: 1969), the "single peroxidase-antiperoxidase (PAP)" (Sternberger LA, Hardy PH Jr, Cuculis JJ, Meyer HG: J Histochem Cytochem 18:315, 1970), the "double PAP" (Vacca LL, Rosario SL, Zimmerman EA, Tomashefsky P, Ng P-Y, Hsu KC: J Histochem Cytochem 23:208, 1975) and the "double bridge" (Ordronneau P, Petrusz P: Am J Anat 158:491, 1980) were compared at both the light and electron microscopic levels. The "double" procedures involved repeating incubations with the bridge antibody, in this case, sheep anti-rabbit gamma globulin, followed either by a second PAP step for the "double PAP" or a second anti-horseradish peroxidase step and a single incubation in horseradish peroxidase for the "double bridge." At both the light and electron microscopic levels the staining intensity was greater with the "double" techniques than with the "single" ones. This is probably due to amplification achieved with the second sheep anti-rabbit gamma globulin step, permitting an increase in the number of horseradish peroxidase molecules bound for each molecule of tissue-bound primary antibody. Also, the quality of the various commercial PAP preparations tested was variable. With the weaker ones the staining intensity could be increased by performing an incubation in fresh horseradish peroxidase after the PAP step. Finally, in electron microscopic studies, the reaction products formed in both the bridge and PAP procedures were identical in shape and size.

scholarly journals Transport into and out of the Golgi complex studied by transfecting cells with cDNAs encoding horseradish peroxidase.

1994 ◽  
Vol 127 (3) ◽  
pp. 641-652 ◽  
Author(s):  
C N Connolly ◽  
C E Futter ◽  
A Gibson ◽  
C R Hopkins ◽  
D F Cutler
Keyword(s):  
Golgi Complex ◽  
Secretory Pathway ◽  
High Sensitivity ◽  
Electron Microscopic Level ◽  
Electron Microscopic ◽  
Double Labeling ◽  
Golgi Stack ◽  
Er Retention ◽  
Peroxidase Cytochemistry ◽  
Golgi Network

We have developed a novel technique with which to investigate the morphological basis of exocytotic traffic. We have used expression of HRP from cDNA in a variety of cells in combination with peroxidase cytochemistry to outline traffic into and out of the Golgi apparatus at the electron microscopic level with very high sensitivity. A secretory form of the peroxidase (ssHRP) is active from the beginning of the secretory pathway and the activity is efficiently cleared from cells. Investigation of the morphological elements involved in the itinerary of soluble ER proteins using ssHRP tagged with the ER retention motif (ssHRPKDEL) shows that it progresses through the Golgi stack no further than the cis-most element. Traffic between the RER and the Golgi stack as outlined by ssHRPKDEL occurs via vesicular carriers as well as by tubular elements. ssHRP has also been used to investigate the trans side of the Golgi complex, where incubation at reduced temperatures outlines the trans-Golgi network with HRP reaction product. Tracing the endosomal compartment with transferrin receptor in double-labeling experiments with ssHRP fails to show any overlap between these two compartments.

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