Hello,
We are interested in visualize actin filaments and microtubules in Leukemia suspension cells. All protocols that I have found are with adherent cells. Has someone optimized the protocol to visualitze actin and microtubules in suspension cells?
Thank you very much for your help.
Andoni
Dear Andoni,
Fluorescently labelled Phalloidin and tubulin antibodies conjugateed with fluorophores are generally the tool of choice for 1 step immunostainings.
The most critical stage is a fast fixation of your samples (typically using buffers containing a % of Glyceraldehyde besides PFA). Using cytoskeleton stabilizers before the fixation may obscure the physiological arrangement in your cells.
How do you wash away the excess of antibody? successive steps of centrifugation and re-suspension in fresh buffer? Something like this might certainly crush the cells and have negative effects in the determination of the cytoskeletal arrange.
Would it be possible to load the fixed cells in a matrix (matrigel,collagen, etc; once fixed the matrix won't influence them) to them perform the immmunostaining? You can later chop the matrix for image acquisition.
Hope it helps,
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