Electron microscopic immunocytochemical localization of substance P in the area postrema of rat

1982 ◽  
Vol 243 (1) ◽  
pp. 141-146 ◽  
Author(s):  
David M. Armstrong ◽  
Virginia M. Pickel ◽  
Donald J. Reis
Author(s):  
Richard W. Burry ◽  
Diane M. Hayes

Electron microscopic (EM) immunocytochemistry localization of the neuron specific protein p65 could show which organelles contain this antigen. Antibodies (Ab) labeled with horseradish peroxidase (HRP) followed by chromogen development show a broad diffuse label distribution within cells and restricting identification of organelles. Particulate label (e.g. 10 nm colloidal gold) is highly desirable but not practical because penetration into cells requires destroying the plasma membrane. We report pre-embedding immunocytochemistry with a particulate marker, 1 nm gold, that will pass through membranes treated with saponin, a mild detergent.Cell cultures of the rat cerebellum were fixed in buffered 4% paraformaldehyde and 0.1% glutaraldehyde (Glut.). The buffer for all incubations and rinses was phosphate buffered saline with: 1% calf serum, 0.2% saponin, 0.1% gelatin, 50 mM glycine 1 mg/ml bovine serum albumin, and (not in the HRP labeled cultures) 0.02% sodium azide. The monoclonal #48 to p65 was used with three label systems: HRP, 1 nm avidin gold with IntenSE M development, and 1 nm avidin gold with Danscher development.


1980 ◽  
Vol 189 (4) ◽  
pp. 721-740 ◽  
Author(s):  
Virginia M. Pickel ◽  
Khushdev K. Sumal ◽  
Susan C. Beckley ◽  
Richard J. Miller ◽  
Donald J. Reis

2003 ◽  
Vol 51 (1) ◽  
pp. 31-39 ◽  
Author(s):  
Toshihiro Takizawa ◽  
Clark L. Anderson ◽  
John M. Robinson

Adequate contrast of ultrathin cryosections is crucial for evaluating morphological detail to assess immunocytochemical localization at the electron microscopic level. We have developed a positive staining method for achieving contrast in ultrathin cryosections, from tissue fixed only in paraformaldehyde, that provides excellent contrast at the electron microscopic level.


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