Effects of acetone, methanol, or paraformaldehyde on cellular structure, visualized by reflection contrast microscopy and transmission and scanning electron microscopy.
The authors recently showed variable subcellular immunoreactivity of the Bcl-2 and Bax proteins after fixation of cell monolayers with acetone, methanol, or paraformaldehyde (PF) followed by methanol (PF/methanol). Here, the authors demonstrate by reflection contrast microscopy and transmission electron microscopy that acetone or methanol fixation result in complete loss of integrity of intracellular structures in contrast with PF or glutaraldehyde fixation. Scanning electron microscopy revealed poor preservation of plasma membrane integrity after fixation in acetone or methanol. Fixation with PF before methanol reduced damage to intracellular and plasma membranes. In addition, Western blot analysis demonstrated loss of Bcl-2 and Bax protein during acetone or methanol fixation, whereas PF fixation before methanol permeabilization markedly reduced this loss. For studies on the intracellular localization of soluble or unknown types of antigen, the authors discourage the use of acetone and methanol as single fixatives.
Hoetelmans
R W
RW
Department of Surgery, Leiden University Medical Center, The Netherlands. Hoetelmans@LUMC.nl
Prins
F A
FA
Cornelese-ten Velde
I
I
van der Meer
J
J
van de Velde
C J
CJ
van Dierendonck
J H
JH
D000096:Acetone / Q000494:pharmacology; D015153:Blotting, Western; D022082:Cellular Structures / Q000187:drug effects* / Q000648:ultrastructure; D005557:Formaldehyde / Q000494:pharmacology; D006801:Humans; D000432:Methanol / Q000494:pharmacology; D008854:Microscopy, Electron; D008855:Microscopy, Electron, Scanning; D008858:Microscopy, Phase-Contrast; D011108:Polymers / Q000494:pharmacology; D011518:Proto-Oncogene Proteins / Q000032:analysis; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000032:analysis; D016707:Tissue Fixation / Q000379:methods / Q000592:standards*; D014407:Tumor Cells, Cultured; D051028:bcl-2-Associated X Protein
C494826:BAX protein, human; D011108:Polymers; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; D000096:Acetone; D005557:Formaldehyde; C003043:paraform; D000432:Methanol