scholarly journals A modification of the unlabeled antibody enzyme method using heterologous antisera for the light microscopic and ultrastructural localization of insulin, glucagon and growth hormone.

1975 ◽  
Vol 23 (9) ◽  
pp. 666-677 ◽  
Author(s):  
S L Erlandsen ◽  
J A Parsons ◽  
J P Burke ◽  
J A Redick ◽  
D E Van Orden ◽  
...  
Keyword(s):  
Growth Hormone ◽  
Gamma Globulin ◽  
Cross Reactivity ◽  
Model Systems ◽  
Electron Microscopic Level ◽  
Immunocytochemical Staining ◽  
Electron Microscopic ◽  
Primary Antiserum ◽  
Enzyme Method ◽  
Unlabeled Antibody

The requirement of using homologous antisera (primary antiserum and peroxidase-antiperoxidase (PAP) complex raised in the same species) in the unlabeled antibody enzyme method has been investigated at the light and electron microscopic level using the localization of insulin, glucagon and growth hormone as model systems. Optimum immunocytochemical staining for all three antigens was observed when sheep or goat antirabbit gamma-globulin (S-ARgammaG or G-ARgammaG) were used to couple rabbit peroxidase-antiperoxidase complex with either guinea pig antisera to insulin (GP-AIS) or glucagon (GP-AGS), or monkey antisera to rat growth hormone (M-ARGH). The cross-reactivity between S-ARgammaG or G-ARgammaG and immunoglobulins in these primary antisera were substantiated by immunoelectrophoresis and radioimmunoassay. S-ARgammaG was shown to produce precipitation arcs with GP-AIS and M-ARGH that were similar to those seen when the latter were reacted with rabbit antiguinea pig gamma-globulin antiserum and goat antimonkey gamma-globulin antiserum, respectively. Radioimmunoassay results revealed that immunoprecipitation of 6-10% as compared to homologous antisera controls yielded excellent staining localization when S-ARgammaG was used for immunocytochemistry. Thus, heterologous antisera (primary antiserum and PAP complex raised in different species) may be used in the unlabeled antibody enzyme method as long as the coupling antiserum shows cross-reactivity with immunoglobulins of the primary antiserum and the PAP complex.

scholarly journals THE UNLABELED ANTIBODY ENZYME METHOD OF IMMUNOCYTOCHEMISTRY QUANTITATIVE COMPARISON OF SENSITIVITIES WITH AND WITHOUT PEROXIDASE-ANTIPEROXIDASE COMPLEX

1974 ◽  
Vol 22 (8) ◽  
pp. 782-801 ◽  
Author(s):  
JOHN P. PETRALI ◽  
DENNIS M. HINTON ◽  
GWEN C. MORIARTY ◽  
LUDWIG A. STERNBERGER
Keyword(s):  
Secretory Granules ◽  
Enzyme Reaction ◽  
Fold Increase ◽  
Immunocytochemical Staining ◽  
Intermediate Lobe ◽  
Reaction Products ◽  
Electron Microscopic ◽  
Specific Staining ◽  
Enzyme Method ◽  
Unlabeled Antibody

Araldite sections of rat pituitary intermediate lobe were used with anti-17-39 adrenocorticotropin in the unlabeled antibody enzyme method to compare electron microscopic immunocytochemical staining by peroxidase-antiperoxidase complex (PAP) with antiserum or purified antibody to peroxidase followed by peroxidase (PO). Quantitation of normalized optical densities of secretory granules offered high significance comparison (P < 0.0001) of experimental with control values and of experimental values with each other. Use of purified antibody (prepared by a new density gradient method) yielded four times higher sensitivity than antiserum to PO, while a 6.5-fold increase would have been expected from the degree of contamination of anti-PO in the serum by nonanti-PO immunoglobulin. Use of PAP was four to five times more sensitive than purified anti-PO and 20 times more sensitive than antiserum to PO. The increased sensitivity of PAP is explained by the high over-all binding affinity of PO for anti-PO in the cyclic PAP molecule, thus preventing the losses of PO that occur during washing when anti-PO and PO have been applied in sequence. Identification of the characteristic, cyclic PAP molecules affords confirmation of specific staining at high resolution. In the sequential application of anti-PO and PO, no PAP molecules are formed, thus making distinction of specific from nonspecific deposition of enzyme reaction products ambiguous. With the use of anti-17-39 ACTH and the intermediate lobe, the unlabeled antibody enzyme method was 16,000-100,000 times more sensitive than radioimmunoassay.

scholarly journals Immunocytochemistry with osmium-fixed tissue. I. Light microscopic localization of growth hormone and prolactin with the unlabeled antibody-enzyme method.

1979 ◽  
Vol 27 (4) ◽  
pp. 867-872 ◽  
Author(s):  
D G Baskin ◽  
S L Erlandsen ◽  
J A Parsons
Keyword(s):  
Growth Hormone ◽  
Epoxy Resin ◽  
Alcoholic Solution ◽  
Immunocytochemical Staining ◽  
Antigenic Determinants ◽  
Fixed Tissue ◽  
Morphological Evaluation ◽  
Enzyme Method ◽  
Thin Plastic ◽  
Unlabeled Antibody

Growth hormone and prolactin were localized on thin plastic sections of rat anterior pituitary gland and mammosomatotropic tumor MtTW15 that were fixed with osmium tetroxide (alone,mixed with aldehydes, or after aldehydes). Intense immunocytochemical staining for both antigens was obtained after plastic was removed from sections with an alcoholic solution of sodium hydroxide. The results indicated that antigenic determinants of rat prolactin and growth hormone were not completely destroyed or inactivated by fixation with osmium and embedment in epoxy resin, and that removal of the polymerized epoxy resin was necessary to obtain light microscopic postembedding immunocytochemical staining of these antigens. The results also demonstrated that tissues which have been conventionally processed for morphological evaluation by electron microscopy may be suitable for postembedding immunocytochemical staining of some antigens for light microscopy.

scholarly journals Unlabeled antibody methods in electron microscopy: a comparison of single and multistep procedures using colloidal gold.

1984 ◽  
Vol 32 (8) ◽  
pp. 799-804 ◽  
Author(s):  
E J Gosselin ◽  
G D Sorenson ◽  
J C Dennett ◽  
C C Cate
Keyword(s):  
New York ◽  
Colloidal Gold ◽  
Single Step ◽  
Electron Microscopic Level ◽  
Step Method ◽  
Electron Microscopic ◽  
Primary Antiserum ◽  
Antigen Localization ◽  
The Individual ◽  
Unlabeled Antibody

A comparative study of five unlabeled antibody methods was conducted on the electron microscopic level using bridging techniques and colloidal gold. The study was based on the principles of the single-step colloidal gold (GLAD) method (Larsson L: Nature 282:743, 1979) and the multistep single- and double-bridge techniques used in postembedding immunoperoxidase procedures (PAP) (Sternberger LA: Immunocytochemistry, 2nd ed. Wiley, New York, 1979). Using medullary thyroid carcinoma and the same lot of primary antiserum (goat anti-calcitonin) for each procedure, it was shown that adequate localization of calcitonin with the single-step GLAD method was attainable only at dilutions of 1:100 or lower. The single-bridge technique using goat anti-calcitonin, sheep anti-goat immunoglobulin (Ig)G, and goat anti-calcitonin and antigen-coated gold, respectively, worked well at dilutions of up to 1:5000 but not at dilutions of 1:10,000, while single- and double-bridging techniques utilizing goat anti-calcitonin, sheep (Sh) anti-goat IgG, and sheep anti-goat IgG-coated gold produced good localization at a 1:10,000 dilution of primary antiserum. A two-step method using goat anti-calcitonin and sheep anti-goat IgG-coated gold, respectively, appeared to be the most sensitive technique, with adequate antigen localization occurring at a dilution of 1:25,000. While in our hands the two-step method appeared superior in sensitivity to the single-bridge IgG-coated gold technique, each method has its own advantages depending on the individual needs of the researcher.

scholarly journals Immunocytochemical localization of renin in the submandibular gland of the mouse.

1978 ◽  
Vol 26 (10) ◽  
pp. 855-861 ◽  
Author(s):  
E Gresik ◽  
A Michelakis ◽  
T Barka ◽  
T Ross
Keyword(s):  
Submandibular Gland ◽  
Adult Mouse ◽  
Secretory Granules ◽  
Microscopic Level ◽  
Electron Microscopic Level ◽  
Light Microscopic Level ◽  
Electron Microscopic ◽  
Granular Convoluted Tubule ◽  
Enzyme Method ◽  
Unlabeled Antibody

Renin was localized in the submandibular gland of the adult mouse at light and electron microscopic levels by the unlabeled antibody enzyme method of Sternberger. At the light microscopic level, renin was confined to the granular convoluted tubule (GCT) segment of the gland with considerable variation among GCT cells in intensity of staining. Some GCT cells failed to stain for renin. The pattern of staining was the same in the gland of male and female mice, but in the glands of females GCT segments were smaller and less numerous. At the electron microscopic level, staining for renin was also confined to the GCT cells, and was localized exclusively to the secretory granules. The intensity of staining of the secretory granules within a given GCT cell varied; some cells contained only minimally reactive or negative secretory granules. All other organelles within the GCT cell, except condensing vacuoles, failed to stain.

scholarly journals ULTRASTRUCTURAL IMMUNOCYTOCHEMISTRY WITH UNLABELED ANTIBODIES AND THE PEROXIDASE-ANTIPEROXIDASE COMPLEX A TECHNIQUE MORE SENSITIVE THAN RADIOIMMUNOASSAY

1973 ◽  
Vol 21 (9) ◽  
pp. 825-833 ◽  
Author(s):  
GWEN C. MORIARTY ◽  
C. MICHAEL MORIARTY ◽  
LUDWIG A. STERNBERGER
Keyword(s):  
Staining Intensity ◽  
Poor Quality ◽  
Immunocytochemical Staining ◽  
Great Promise ◽  
Electron Microscopic ◽  
Immunocytochemical Technique ◽  
Titration Curves ◽  
Rat Pituitary ◽  
Normal Rat ◽  
Unlabeled Antibody

Titration curves were developed with antisera to 17-39ACTH (adrenocorticotropin) and 1-39ACTH with the techniques of radioimmunoassay and electron microscopic immunocytochemistry. For the latter method, the unlabeled antibody peroxidase-antiperoxidase complex immunocytochemical technique was used to stain normal rat pituitary intermediate lobes. By radioimmunoassay standards, the 17-39ACTH antiserum was of poor quality. Its titration curve exhibited a flat slope and it did not bind a significant amount of labeled antigen beyond a 1: 30 dilution. However, immunocytochemical staining was detected with this antiserum at dilutions as high as 1:1,500. The antiserum to 1-39ACTH was of better quality by radioimmunoassay standards. It bound 45% of the labeled antigen at a dilution of 1:5,000. Immunocytochemical staining intensity was nearly maximal at a 1:5,000 dilution and decreased progressively to a limiting value at 1:16,000. However, when incubation times in the antisera were increased from 3 min to match those of the radioimmunoassay (48 hr) maximal staining was achieved at dilutions as great as 1:512,000 where only trace amounts of the labeled antigen were bound in the radioimmunoassay. It was concluded that the unlabeled antibody peroxidase-antiperoxidase complex immunocytochemical technique was sensitive enough to detect antibodies in sera of low titer and/or avidity which are not detected by a radioimmunoassay. The technique holds great promise for a sensitive assay system.

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 gamma-Aminobutyric acid-containing terminals can be apposed to glycine receptors at central synapses

1987 ◽  
Vol 104 (4) ◽  
pp. 947-956 ◽  
Author(s):  
A Triller ◽  
F Cluzeaud ◽  
H Korn
Keyword(s):  
Gaba Receptors ◽  
Glycine Receptor ◽  
Cross Reactivity ◽  
Aminobutyric Acid ◽  
Electron Microscopic Level ◽  
Acid Decarboxylase ◽  
Gamma Aminobutyric Acid ◽  
Glycine Receptors ◽  
Electron Microscopic ◽  
Double Labeling

The distributions of terminals containing gamma-aminobutyric acid (GABA) and of endings apposed to glycine receptors were investigated cytochemically in the ventral horn of the rat spinal cord. For this purpose, a polyclonal antibody raised to recognize glutamic acid decarboxylase (GAD), a synthetic enzyme for GABA, and three monoclonal antibodies (mAb's) directed against the glycine receptor were used. Double immunofluorescence showed that, surprisingly, GAD-positive terminals are closely associated in this system with glycine receptors at all the investigated cells, most of which were spinal motoneurons. Furthermore, double labeling was performed with immunoenzymatic recognition of GAD and indirect marking of mAb's with colloidal gold. With this combined approach, it was found, at the electron microscopic level, that all GAD-positive terminals are in direct apposition with glycine receptors while, on the other hand, not all glycine receptors are in front of GABA-containing boutons. This result is not due to a cross-reactivity of mAb's with GABA receptors as shown by using as a control synapses known to use GABA as a neurotransmitter in the cerebellar cortex. Indeed, no glycine receptor immunoreactivity was detected on Purkinje cells facing basket axon terminals. However, Purkinje neurons can express glycine receptor immunoreactivity at other synaptic contacts. Assuming that the presence of postsynaptic receptors for glycine indicates that this amino acid is used for neurotransmission at a given synapse, our results strongly support the notion that GABA and glycine, two classical inhibitory transmitters, coexist at some central connections. However, such is not always the case; in the cerebellum, Golgi terminals impinging on the dendrites of granule cells are either GAD-positive or face glycine receptors, in a well-segregated manner.

Correlative histochemical and immunocytochemical techniques for the study of classical transmitters and neuropeptides

1989 ◽  
Vol 47 ◽  
pp. 902-903
Author(s):  
M.T. Pelto-Huikko
Keyword(s):  
Phosphate Buffer ◽  
Mammalian Species ◽  
Microscopic Level ◽  
Biologically Active ◽  
Trophic Factors ◽  
Electron Microscopic Level ◽  
Immunocytochemical Staining ◽  
Electron Microscopic ◽  
Endocrine Organs ◽  
Catecholamine Fluorescence

A growing number of small biologically active peptides has been discovered in the nervous system and the endocrine organs of different mammalian species. These so-called neuro- or regulatory peptides may function as neurotransmitters, hormones or trophic factors. In many cases neuropeptides are colocalized in the same neurons and endocrine cells with classical transmitters and hormones. To understand the functions of neuropeptides it is important to determine the exact localization of neuropeptides in tissues and to clarify their relations to classical transmitters. In this paper a method for correlative visualization of epinephrine and norepinephrine with formaldehyde-glutaraldehyde (Faglu) fluorescence and subsequent demonstration of neuropeptides at light and electron microscopic level with pre-embedding immunocytochemistry in the same Vibratome section is described. A method for correlative demonstration of acetylcholinesterase (AChE) and neuropeptides at light microscopic level is also presented.Adrenal glands were fixed with a mixture of 4% paraformaldehyde and 0.2% glutaraldehyde in 0.1M phosphate buffer, pH = 7.3. The adrenals of small rodents such as rat and hamster were fixed by immersion fixation for 6 h. The adrenals of larger species like cats and guinea pigs were fixed by perfusion for 20 min and further fixed by immersion for 1 hr. After fixation the tissues were rinsed several times in phosphate buffer and 7- to 10-um-thick sections were cut with a Vibratome. Sections are embedded in 0.5M Tris-saline buffer, pH = 7.6, which contained 20% glycerol and the catecholamine fluorescence was photographed (Fig. 1). After photography the coverslip was removed and the sections were processed free-floating through the steps of the pre-embedding modification of the PAP-method. After immunocytochemical staining the sections were post-fixed with 2% aqueous osmiumtetroxide for 1 h and dehydrated in graded ethanol series and flat-embedded in Epon under plastic coverslip. The areas photographed for catecholamine fluorescence were identified and photographed (Fig. 2). Desired areas were processed for electron microscopic examination. Human sympathetic ganglia were obtained from surgery and fixed with 4% paraformaldehyde for 6 h. Tissues were cryoprotected with 10% sucrose and 5 um frozen sections were cut on glass slides. Immunocytochemistry was carried out with indirect immunofluorescence method. After immunocytochemistry the sections were reacted for AChE according to the method of Karnovsky and Roots. Rhodamine-fluorescence and AChE were photographed using appropriate filters.

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