ISSN:
0741-0581
Keywords:
Immunocytochemistry
;
Cell culture
;
Cytoskeleton
;
Scanning electron microscopy
;
Life and Medical Sciences
;
Cell & Developmental Biology
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Natural Sciences in General
Notes:
This technique permits the visualization of microtubules in situ by employing silver-enhanced immunogold labeling and backscattered electron imagery. For best results, monolayer cultures of PtK2 cells are lysed with Triton X-100 in a microtubule stabilizing buffer, fixed with 1% glutaraldehyde, reduced with NaBH4, incubated with monoclonal antitubulin and 5-nm gold-labeled anti-IgG, silver enhanced, freeze dried, lightly coated with aluminum, and examined in an SEM equipped with a backscattered electron detector. A high contrast view of the entire microtubule complex of each cell is obtained. Microtubules in freeze-dried preparations have relatively smooth surfaces, whereas those in critical point dried preparations are more irregular or beaded. At high magnifications, an unstained inner core of each microtubule can be resolved. Backscattered electron imaging appears to be a promising technique for localizing cytoskeletal proteins and other intracellular antigens that can be labeled with immunogold and enhanced with silver.
Additional Material:
20 Ill.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1002/jemt.1060050306
Permalink
