Rapid microwave fixation of cell monolayers preserves microtubule-associated cell structures.
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 MW fixation 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 showing its suitability at the fine structural level. Here, we provide a rapid MW fixation protocol for cell monolayers on a subminute time scale. The impact of the MW-accelerated glutaraldehyde fixation on temperature-sensitive cytoskeletal components such as microtubules was evaluated. For testing the effectiveness of MW-assisted primary fixation, saponin treatment of the monolayers was 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 MW fixation with automatic processing in a tissue processor is provided.
Reipert
Siegfried
S
Department of Molecular Cell Biology, Max F. Perutz Laboratories, Dr. Bohr-Gasse 9, University of Vienna, A-1030 Vienna, Austria. siegfried.reipert@univie.ac.at
Kotisch
Harald
H
Wysoudil
Bhuma
B
Wiche
Gerhard
G
D000818:Animals; D002502:Centrioles / Q000648:ultrastructure; D004847:Epithelial Cells / Q000166:cytology* / Q000648:ultrastructure; D004853:Epoxy Resins; D005404:Fixatives; D005976:Glutaral; D007614:Macropodidae; D008870:Microtubules / Q000648:ultrastructure*; D008872:Microwaves; D015388:Organelles / Q000648:ultrastructure; D012503:Saponins; D016610:Tissue Embedding; D016707:Tissue Fixation / Q000379:methods*
D004853:Epoxy Resins; D005404:Fixatives; D012503:Saponins; D005976:Glutaral