Staining for F-actin (phalloidin)

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Guy Sovak
Guy Sovak's picture
Staining for F-actin (phalloidin)

The protocol is taken from the Invitrogen / Molecular probe web site.
(check for protocol section)

The protocol was tryed by me and I had excelent results.

1 Wash cells twice with prewarmed phosphate-buffered saline, pH 7.4 (PBS).
2 Fix the sample in 3.7% formaldehyde solution in PBS for 10 minutes at room temperature.
Note: Methanol can disrupt actin during the fixation process. Therefore, it is best to avoid
any methanol containing fixatives. The preferred fixative is methanol-free formaldehyde.
3 Wash two or more times with PBS.
4 Place each coverslip in a glass petri dish and extract it with a solution of acetone at ?20°C or
0.1% Triton X-100 in PBS for 3 to 5 minutes.
5 Wash two or more times with PBS.
6 When staining with any of the fluorescent phallotoxins, dilute 5 ?L methanolic stock solution
into 200 ?L PBS for each coverslip
to be stained. To reduce nonspecific background staining
with these conjugates, add 1% bovine serum albumin (BSA) to the staining solution.
It may
also be useful to pre-incubate fixed cells with PBS containing 1% BSA or with the Image-iT
FX signal enhancer (I36933) for 2030 minutes prior to adding the phallotoxin
staining solution.
When staining with biotin-XX phalloidin (B7474), dilute 10 ?L of the methanolic stock solution
into 200 ?L PBS for each coverslip to be stained.
When staining more than one coverslip, adjust volumes accordingly. For a stronger signal, use
2 or 3 units per coverslip.
7 Place the staining solution on the coverslip for 20 minutes at room temperature (generally,
any temperature between 4°C and 37°C is suitable). To avoid evaporation, keep the coverslips
inside a covered container during the incubation.
8 Wash two or more times with PBS.
9 When using biotin-XX phalloidin, incubate for 30 minutes with 100 ?L of a 10 ?g/mL solution
of fluorescent or enzyme-conjugated
streptavidin dissolved in 100 mM Tris-HCl, pH 7.5,
150 mM NaCl, 0.3% Triton X-100 and 1% BSA. Incubate for 15 minutes at room temperature.
After incubation, wash the coverslip with PBS. To develop enzyme activity, follow a procedure
recommended for the specific enzyme.
10 For long-term storage, the cells should be air dried and then mounted in a permanent
mountant such as ProLong Gold reagent or Cytoseal. Specimens
prepared in this manner.

retain actin staining for at least six months when stored in the dark at 26°C

labovich's picture

i stained according to that protocol primary fibroblasts after i calibrated the system. in some of the cells i got a positive stain in the nucleus. i didn't use DMSO or methanol.
can that be an artifact?
i know that f-actin can be in the nucleus but i don't think that that's the actin that phalloidin can stain.

Guy Sovak
Guy Sovak's picture
Hi ,

Hi ,
It could be an artifact but I would need to see the pic so it would be the best if you could attach it. If yyou could send a negative control . do you have the nucleus stained with DAPI?

furein's picture
I've the same problem and the

I've the same problem and the attached picture shows the nuclear staining in HUVEC cells.

labovich's picture

actually, i finished my thesis 6 monhs ago...
but- the positive staining in the nucleus was due to a micoplasm infection!!

Guy Sovak
Guy Sovak's picture
It may be a contamination.

It may be a contamination.
Usually in Micoplasma contamination you will have many more small blue dots surrounding the nucleus.

actin_network's picture


To stain actin the protocol I am following.

cells in coverslip. washing 2 times in PBS

Add formaldehyde 10%  for 20 mins

wash in PBS 2 times

triton 0.2 % for 15 to 20 mins

adding 100PBS+ 2µl of phalodin stock for 40mins to 50mins

wash 2 times

fluosheild overnight

my nucleus get stained very well but actin doesnot get stained ??
i am not understanding !!!