Fixation method for phalloidin staining - (Jul/28/2005 )

Hi,

I've just done my first IF experiment so I'm very new to this field.
I'm now going to do a double labeling experiment on actin and my protein of interest. I've just ordered Alexa 488 phalloidin for staining actin.
The thing I'm wondering about is fixation. Indeed, I've used the methanol fixation method for my first experiment, and I've read that this method is not suitable for phalloidin. Is that absolute or will it work anyway but not as good as after formaldehyde fixation ? Is it worth trying ?

The resasn for this question is that a guy in my lab keeps tellig me that the FLAG antibody (FLAG M2 mouse antibody from Sigma) that I've used in my first experiment for my favourite protein does not work with formaldehyde fixation... Do you have any experience with it ??

Thanks.

Anne

We use 10% formaldhyde for fixing followed by 5 minutes with just Methanol for phalloidin. It seems to work well and we can look at other proteins in these cells but we haven't used the FLAG antibody before. Hopefully, this is of some help.

Thanks.
I think I'll go for formaldehyde fixation then, I've found many papers where they used it successfully with FLAG antibody.
What i'm wondering about now is if I'd better to add the phalloidin with my secondary antibody or to do an additional incubation step afetr washing out the secondary antibody (I found both methods).
What's your experience with that ? What dilution of phalloidin (Alexa 488 phalloidin from Molecular probes for me) are you using ??

Anne

Sorry I made a mix up and it should be cold acetone and not methanol...that is what I get for doing it from memory rather than looking at the procedure.

Add ICE COLD Acetone to permeabilize cells. Place at -20°C for 5 min.
Remove Acetone and wash 2X w/ PBS
Add 200uL of blocking solution to each chamber. Incubate 1 hr at RT or O/N at 4°C.
Aspirate and add 200uL fluorescent phalloidin (5uL + 200uL blocking).
Incubate 30 - 60min, RT.
Incubate other primary abs for1hr at RT followed by secondary for 1 hr

We use the same stain as you for phalloidin