Staining for F-actin (phalloidin)

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Guy Sovak
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Staining for F-actin (phalloidin)

The protocol is taken from the Invitrogen / Molecular probe web site.
https://catalog.invitrogen.com/index.cfm?fuseaction=viewCatalog.viewProductDetails&productDescription=4179&catname=North%20America%20Main
(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
labovich's picture
hi

hi
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
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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?
Guy

furein
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I've the same problem and the

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

labovich
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tanks

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

Guy Sovak
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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.
Guy

actin_network
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Hi,

Hi,

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 !!!