Comparison of cryofixation and aldehyde fixation for plant actin immunocytochemistry: Aldehydes do not destroy F-actin

For walled plant cells, the immunolocalization of actin microfilaments, als o known as F-actin, has proved to be much trickier than that of microtubule s. These difficulties are commonly attributed to the high sensitivity of F- actin to aldehyde fixatives. Therefore, most plant studies have been accomp lished using fluorescent phallotoxins in fresh tissues. Nevertheless, conce rns regarding the questionable ability of phallotoxins to bind the whole co mplement of F-actin necessitate further optimization of actin immunofluores cence methods. We have compared two procedures: (1) formaldehyde fixation a nd (2) rapid freezing and freeze substitution (cryofixation), both followed by embedding in low-melting polyester wax. Actin immunofluorescence in sec tions of garden cress (Lepidium sativum L.) root gave similar results with both methods. The compatibility of aldehydes with actin immunodetection was further confirmed by the freeze-shattering technique that does not require embedding after aldehyde fixation. It appears that rather than aldehyde fi xation, some further steps in the procedures used for actin visualization a re critical for preserving F-actin. Wax embedding, combined with formaldehy de fixation, has proved to be also suitable for the detection of a wide ran ge of other antigens.