cytoskeleton staining

[Mary Eua]

hello everybody,
Has anyone tried actin or ezrin or vimentin staining for confocal microscopy in T cells? I 've read that methanol fixation could cause some problems with the cytoskeleton integrity. What do u suggest I should try for start anyway (talking about for both fixation and permeabilization)? Do u think I need to use some kind of cytospin since T cells do not attach to the plate? I would prefer to do the staining having cells in suspension and then apply them later on a microscopy slide.Thanks a lot guys!

[Jon Moulton]

Hi Mary,

I work for Gene Tools. This describes a method using one of my company's products.

We tried staining actin using an anti-actin antibody fragment (50kD) labelled with Texas Red and delivered using the Endo-Porter delivery reagent. The cells we imaged were live. The cytoskeletal staining looked very nice through the scope. This offers a method for cytoskeletal imaging with minimal perturbation of the cytoskeleton.

Cells vary in their tolerance to Endo-Porter; for most cell types there is a concentration window allowing delivery without significant toxicity. We used HeLa cells.

Here is a description of Endo-Porter: http://www.gene-tools.com/node/24

Uptake occurs by endocytosis, so you would put the cargo (fluorescent antibody fragment) and the Endo-Porter onto your cells and let the mixture incubate overnight, then wash the cells and observe them the next day. You could deliver to the cells in suspension overnight, transfer them to a surface to which they will adhere and then gently wash then before observing. However, uptake is a bit better when cells are adherent (we think Endo-Porter complexes might be settling out of solution onto adherent cells).

Let me know if I can help.

- Jon

[Viper]

I have stained both for actin and ezrin, but in epithelial cells (MDCK cells).

If you'd like to know more just let me know.

The ezrin antibody I used is a mAb from BD Translabs, works for Westerns, IPs, and immuno too. Pretty hard to find an antibody that can do all 3.

[amit.das]

Hi Mary,

I do regularly stain actin cytoskeleton in fixed cells. Though I have not ever done on T-Cells but till date I have tried on HeLa, NIH3T3, HEK293 and N2A cells and also in some phytoplankton (with a collaborator group of our lab). I always fix cells with 4% freshly prepared para-formaldehyde (Sigma) and stain in either Rhodamine-Phalloidin (Molecular Probes) or Alexa 488 - Phalloidin (Molecular Probes) as per the protocol mentioned in manufacturer website. A 100 fold more dilution that the company recommend also worked fantastically. Post staining I wash twice with 1X PBS and then mount. It did not ever require me to permialize cells but this step might be dependent on your application.

This procedure worked amazing for me. Wish it would be of some help to you.

Cheers,

Amit Das,
SRF-CSIR,
Dept. of Biological Sciences,
IISER-Kolkata.
India.

[Mak86]

I routinely stain T cells for Actin in our lab.
Fixation and Permebilization is important for staining t cell cytoskeletal components.
I use Methanol free 4% formaldehyde in PBS made up from 16% Formaldehyde ampules(Fisher PN28908).10min at room temp does the job for fix. After this I wash a few times with PBS.
For Actin, i assume you would be using the phalloidin conjugate dyes which bind to F actin. I've never tried the anti-Actin antibodies since this dye works so well for F actin.
For Permeabilization, I use 0.1% triton X-100 in pbs (sigma) for 5minutes and then wash 3X with PBS.

Mak

[dane_hg]

I dont know about cytospin but I can tell you that no antibody is a match for Phalloidin because a toxin is, by definition, much more specific than an antibody.
I used 4% Parafolmaldehyde in PBS for fixation (10min @ 37°C is a bit better than the classical 15min RT°). Methanol is not very compatible with actin!
Permeabilize with 0.1%Triton in PBS.
No need to wash.
Apply Phalloidin conjuagted with a fluorochrome (Molecular Probes) diluted 1:100 for 30min.
Mount and you're good to go.

Cheers