alternatively recombinant expression of AFP-labeled actin via baculovirus-transduction-available from molecular probes. or: Microinjection :-)

josef

BacMam 2.0 from invitrogen. This is insect virus based fluorescent protein dye family. It doesn't have mammalian promoters, and doesn't replicate in mammalian cells. Another option could be microinjection of fluorescently labeled G-Actin in every cell you wanna stain.

Besides the obvious use of the various available GFP-constructs you can microinject rhodamine-labeled actin. However you will not find any vital stains

In some cases phalloidin worked in living plant cells, but I don't have experience for mammalian cells. Did you think about GFP-based constructs?

Hi,

I have a lifeact-SNAP-tag construct can be label without fixation. SNAP-tag can be label by peamable dyes for 30 min.

Rhodamine-labeled phalloidin does the trick for many years in unfixed plant cells (like onion epidermal peels) in our practical courses. The procedure is explained in Verbelen et al. (2001) The onion and the student, a fruitful combination! Journal of Biological Education 35, 196-200.

Be aware that it takes 30-45 minutes before actual staining is visible and that the cells don't like it for a long time, so they will die....

Lifeact-GFP is available in yeats and mouse. Really depends on your species? alternatively labelling actin binding proteins is anotheri indirect way. E.g Abp140-GFP or tropomyosin etc. good luck!

you can transfect your cells with lifeact-GFP plasmid and rely on the expression of the probe in the cell to stain F-actin.

Lifeact SNAP-tag can be label by organic dyes which is more photostable than fluorescent proteins like as GFP.

LifeAct or Snap-tags!

Also LifeAct!

The real "Actin-GFP" constructs can some how interfere with the actin polymerisation (and might change the physical chemistry of the actin polymerisation) and are the older method. Nevertheless they are still fine and accepted for publication.

Thank you all for your answers so far. I want to clarify what I want to do:

The limiting factor in my case is not a difficulty in transfection. Rather, I am trying to set up a High throuput microscopy screen looking at many different cell lines with many different perturbations. I am no even interested in keeping the cells alive, it's just that some of the other stains require membranes to remain intact (e.g. mitochondria stains) and ideally in such a screen one wants to keep handling of the cells (fixation, washing, permeabilizatio, ...) to a minimum.

It is a very good question. I was wondering the same thing about microtubules. What would you recommend for observing microtubules in unfixed cells?

Thank you.

As other people suggest, LifeAct is working well for Actin. GFP-tubulin and GFP-intermidiate filamet subunit (vimentin, keratin, neurofilament etc.) are also able to assemble to the filamentous form. Of course, they are visible in the living cell well and you can observe that they are transported in the cell too. But I have to say that the conjugate site is sometimes very important for the assemble. When you use them, you should confirm which N-terminal or C-terminal is better for you purpose. Almost all fluorescent protein, mChery, Eos(photo convertible fluorescence protein), photo-covertible GFPwell are working well.

I have tried different actin-XFP proteins (including LifeActin -GFP and -KO) but, as others have pointed out before, one of the problems I have found is that the over-expression of many of these tag-protein in my system generate dominant and aberrant phenotypes. Finally I have found two very good options: Utrophin-GFP and Moesin-GFP. They work pretty well regarding fluorescence intensity and stability (so in-vivo it will be easy for you to visualise them even if that was not your first objective), and they don't exhibit any defective phenotype when over-expressed in my system. Good luck!

In plant cells we regularly use rhodamin-labeled phalloidin without fixation. Protocol can be found here: Verbelen et al. (2001) The onion and the student, a fruitful combination! Journal of Biological Education 35, 196-200. You could try with different amounts of NP-40 to permeabilise the membrane to a lesser extent. Good luck!

you can also try Chromobodies http://www.chromotek.com/products/chromobodies/actin-chromobodyr/

Fluo-labeled taxol from Mol Probes exists. Check the details in Mol Probes Handbook.

http://www.lifetechnologies.com/hu/en/home/references/molecular-probes-the-handbook.html

For live cell, actin probe phalloidin looks big to penetrate.

Hello,

I'm asking here, beacuse you guys are expert in this "actin" topic....I also use lifeact rfp (it works very well when levels are not too high), but now i'm trying to clone it in another vector, but given that it was a gift, i cannot find the map..

I know that it is in pegfp n1, have you ever tried to digest it? Do you know how it is cloned in the vector?

thanks a lot

Hi,

we recently developed in my Lab a new probe that stains F-actin selectively. It works with fixed samples but most importantly with live specimens. if anyone is interested here is the paper

Fluorogenic probes for live-cell imaging of the cytoskeleton'

http://www.nature.com/nmeth/journal/v11/n7/full/nmeth.2972.html:

The probes are commercially available here:

http://spirochrome.com/

Best

Luc