In the literature, I have found at least 10 different (or rather, only marginally similar) methods for plant (leaf) samples fixing (formaldehyde/glutaraldehyde) and subsequent phalloidin stain for actin. Do you use any standardized protocol for this technique? If so, what are the details? What plants is it good for?
Dear Marcin,
Our protocol works very well with maize, wheat and arabidopsis. In case you work only for actin staining and you observe with a CLSM, things are simple as you don't need any enzyme step. Just apply MBS (see the protocol in the Panteris et al. 2006 attached) before mild fixation with PFA. If you work with chlorotic plants or you can focus on epidermal cells only, a "red" phalloidin (alexa 568 or tritc-phalloidin) is brilliant. If you expect to study cells with chlorophyll, then try a "green" fluorophore equivalent. In case you work with conventional fluorescence microscopy (not CLSM) you probably have to separate cells. Then try a mild digestion with 1-2% macerozyme and macerate your sample through a Pasteur pipette. For optimum staining add also 1% (v/v) phalloidin in the enzyme mixture. Be gentle for actin and the result will be satisfying. Do not overstabilize with MBS to avoid any artifacts in actin bundling. Good luck!
Manolis
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