Rapid Microwave Fixation of Cell Monolayers Preserves Microtubule-associated Cell Structures
Siegfried Reipert 1*, Harald Kotisch 1, Bhuma Wysoudil 1 and Gerhard Wiche 1
1 Department of Molecular Cell Biology, Max F. Perutz Laboratories, University of Vienna, Vienna, Austria
* To whom correspondence should be addressed. E-mail: siegfried.reipert{at}univie.ac.at .
Submitted on October 20, 2007
Accepted on 2 April 2008
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Abstract |
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Microwave (MW) fixation has been suggested as a method to rapidly immobilize cellular dynamics for fine structural studies in the electron microscope. To show its suitability for studies on cell monolayers, one has to apply it systematically in correlation with samples on the light microscopy level. Examples for MW fixation of cell monolayers, however, are still rare. MW-accelerated fixation for relatively long periods of time (1-2 min) has been reported without demonstrating its suitability at the fine structural level. Here, we provide a rapid MW fixation protocol for cell monolayers on a sub-minute time scale. The impact of the MW-accelerated glutaraldehyde fixation on temperature-sensitive cytoskeletal components such as microtubules has been evaluated. For testing the effectiveness of MW-assisted primary fixation, saponin treatment of the monolayers has been included. Simultaneous MW-accelerated fixation and extraction by saponin was necessary to achieve a gradual improvement in visualization of cytoskeletal aspects in association with cell junctions, mitochondria, and centrioles. To establish a valuable routine program for fine structural studies of resin-embedded cell models on substrata, a protocol combining of MW fixation with automatic processing in a tissue processor is provided.
Key Words:
microwave fixation, cell monolayers, saponin extraction, electron microscopy, microtubules, cell junctions
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