Co-localization of chromogranin A and B, secretogranin II and neuropeptide Y in chromaffin granules of rat adrenal medulla studied by electron microscopic immunocytochemistry

1989 ◽  
Vol 91 (6) ◽  
pp. 473-477 ◽  
Author(s):  
H. -J. Steiner ◽  
K. W. Schmid ◽  
R. Fischer-Colbrie ◽  
G. Sperk ◽  
H. Winkler
Keyword(s):  
Neuropeptide Y ◽  
Adrenal Medulla ◽  
Chromogranin A ◽  
Chromaffin Granules ◽  
Electron Microscopic ◽  
Electron Microscopic Immunocytochemistry ◽  
Secretogranin Ii

Artifact-free electron microscopic immunocytochemistry on rat brain tissue

1991 ◽  
Vol 49 ◽  
pp. 272-273
Author(s):  
Veronika Burmeister ◽  
N. Ludvig ◽  
P.C. Jobe
Keyword(s):  
Microscopic Examination ◽  
Free Electron ◽  
Electron Microscopic Examination ◽  
Ultrastructural Localization ◽  
Rabbit Serum ◽  
Electron Microscopic ◽  
Electron Microscopic Immunocytochemistry ◽  
Phosphate Buffered Saline ◽  
Rat Brain Tissue ◽  
Immune Rabbit

Electron microscopic immunocytochemistry provides an important tool to determine the ultrastructural distribution of various molecules in both normal and pathologic tissues. However, the specific immunostaining may be obscured by artifactual immunoreaction product, misleading the investigator. Previous observations show that shortening the incubation period with the primary antibody from the generally used 12-24 hours to 1 hour substantially reduces the artifactual immunostaining. We now extend this finding by the demonstration of artifact-free ultrastructural localization of the Ca2/calmodulindependent cyclic nucleotide phosphodiesterase (CaM-dependent PDE) immunoreactivity in brain.Anesthetized rats were perfused transcardially with phosphate-buffered saline followed by a fixative containing paraformaldehyde (4%) and glutaraldehyde (0.25%) in PBS. The brains were removed, and 40μm sections were cut with a vibratome. The sections were processed for immunocytochemistry as described by Ludvig et al. Both non-immune rabbit serum and specific CaM-dependent PDE antibodies were used. In both experiments incubations were at one hour and overnight. The immunostained sections were processed for electron microscopic examination.

Immunogold labeling of the calcium binding protein synexin in isolated adrenal chromaffin granules and chromaffin cells

1990 ◽  
Vol 48 (3) ◽  
pp. 952-953
Author(s):  
Gemma A.J. Kuijpers ◽  
Harvey B. Pollard
Keyword(s):  
Adrenal Medulla ◽  
Calcium Binding ◽  
Chromaffin Cells ◽  
Cell Activation ◽  
Structural Information ◽  
Dependent Manner ◽  
Chromaffin Granules ◽  
Immuno Electron Microscopy ◽  
Ultrathin Sections ◽  
Adrenal Chromaffin

Exocytotic fusion of granules in the adrenal medulla chromaffin cell is triggered by a rise in the concentration of cytosolic Ca2+ upon cell activation. The protein synexin, annexin VII, was originally found in the adrenal medulla and has been shown to cause aggregation and to support fusion of chromaffin granules in a Ca2+-dependent manner. We have previously suggested that synexin may there fore play a role in the exocytotic fusion process. In order to obtain more structural information on synexin, we performed immuno-electron microscopy on frozen ultrathin sections of both isolated chromaffin granules and chromaffin cells.Chromaffin granules were isolated from bovine adrenal medulla, and synexin was isolated from bovine lung. Granules were incubated in the presence or absence of synexin (24 μg per mg granule protein) and Ca2+ (1 mM), which induces maximal granule aggregation, in 0.3M sucrose-40m MMES buffer(pH 6.0). Granules were pelleted, washed twice in buffer without synexin and fixed with 2% glutaraldehyde- 2% para formaldehyde in 0.1 M phosphate buffer (GA/PFA) for 30 min. Chromaffin cells were isolated and cultured for 3-5 days, and washed and incubated in Krebs solution with or without 20 uM nicotine. Cells were fixed 90 sec after on set of stimulation with GA/PFA for 30 min. Fixed granule or cell pellets were washed, infiltrated with 2.3 M sucrose in PBS, mounted and frozen in liquid N2.

Ultrastructural Immunocytochemistry of Five Rat Pituitary Adenomas

1980 ◽  
Vol 38 ◽  
pp. 724-725
Author(s):  
D. J. McComb ◽  
N. Ryan ◽  
E. Horvath ◽  
K. Kovacs ◽  
E. Nagy ◽  
...  
Keyword(s):  
Pituitary Adenomas ◽  
Pituitary Tumors ◽  
Electron Microscopic ◽  
Electron Microscopic Immunocytochemistry ◽  
Transmission Electron ◽  
Rat Pituitary ◽  
Radiation Induced ◽  
Group 5 ◽  
Group 2 ◽  
Microscopic Techniques

Conventional light and electron microscopic techniques failed to clarify the cellular composition and derivation of spontaneous and induced, intrasellar and transplanted pituitary adenomas in rats (1). In the present work, electron microscopic immunocytochemistry was applied to evaluate five adenohypo-physial tumors using a technique described by Moriarty and Garner (2). Spontaneously occurring pituitary adenomas (group 1) were harvested from aging female Long-Evans rats. R-Amsterdam rats were treated with 2 x 1.0 mg estrone acetate (HogivaI) s.c. weekly for 6 months. Pituitary adenomas in excess of 30 mg were removed from these animals to make up the tumors of group 2. Groups 3 and 4 consisted of estrogen-induced autonomous transplan¬ted pituitary tumors MtT.WlO and MtT.F4. Group 5 was a radiation-induced transplanted autonomous pituitary tumor MtT.W5. The tumors of groups 3,4 and 5 were allowed to proliferate in host rats 6-8 weeks prior to removal for processing. Tissue was processed for transmission electron microscopy (glutaraldehyde fixation, OsO4 postfixation and epoxy resin embedding), and electron microscopic immunocytochemistry (3% paraformaldehyde fixation and Araldite embedding).

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