Dear all,
does anybody have experience with the simultaneous fixation, permeabilization and Phalloidin staining of adherent cells (fibroblast or endothelial cells/cell lines)? Many companies suggest the following protocol:
Simultaneous Fixation, Permeabilization, and Fluorescent Phallotoxin Staining
The phallotoxins appear to be stable for short periods in 4% formaldehyde fixation buffers. This permits a rapid one-step fixation, permeabilization, and labeling procedure as follows.
1) Prepare a 1 mL solution containing 50 to 100 µg/mL lysopalmitoylphosphatidylcholine and 3.7% formaldehyde and then add 5–10 units of fluorescent phallotoxin (approximately 25 to 50 µL of methanolic stock solution).
2) Place this staining solution on cells and incubate for 20 minutes at 4°C.
3) Rapidly wash three times with buffer.
4) Mount coverslips and view.
Has anybody tried it? Lysopalmitoylphosphatidylcholine is quite expensive. I am wondering whether Triton X100 will lead to the same result.
Many thanks
Valeria
Dear Valeria, I have experienced the staining of phalloidin on endothelial cells (both murine aortic ECs, human aortic and mutated HUVEC, named Eahy). From my personal experience, only murine aortic ECs required a pre-coating of the wells (I used both Transwell filters, glasses and plastic 8-well chamber slides). In general, I washed once the cells with cold-PBS containing Calcium and Magnesium (to preserve cell-cell connection), then I fixed the cells for 30 min at room temperature with 2% PFA (prepared in PBS with salts), then three washing steps, then 30 min at room temperature with 0.1% Triton x-100 (diluted in PBS with salts). I did not block the cells but you could by using PBS/BSA 1% for 30 min. I incubated the cells with phalloidin diluted in PBS/BSA 1% for 45 minutes. Then, I used a mounting media (Prolong or Vectashield) and accordingly I have acquired the pictures. From my personal experience the protocol worked very well, without using particular buffers, but I made the incubation after the fix/perm.
Alternatively, I tried (and it worked very well) the BD cytofix/cytoperm (BD Biosciences, Catalog No.554714) that allow you to fix and permeabilyze at the same time. Moreover, when you remove the buffer the cells are no longer permeabilized. It is usually used for FACS purposes.
Hope it will be helpful! And if you need additional information just let me know.
I wish you a nice day!
Lucia
HI this is the method I used.
1. Fix cells with 90% MethOH (10mins)
2. Wash with PBS x2
3. Add 0.1% triton X to each sample for 4-6 mins
4. Wash x2 PBS
5. Add working stock of Phalloidin and incubate for 3 hrs 37oC
6. Wash x2 PBS
7. Counter stain 1:500 DAPI for 5 mins
8. Wash x3 PBS.
9. Mount onto cover slip and image.
Dear Lucia,
we have a good protocol for phalloidin-staining. I am interested now in a One Step-Phalloidin staining protocol. The BD cytofix/cytoperm-solution sounds good. Do you like to share your experience with it with regard to phalloidin staining of adherent cells?
Many thanks
Valeria
Why don't you try to compare the two method? I guess choline is a weak permeabilizer. Try digitonine too. If you have time. Bests!
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