Hi:
We would like to stain the cancer cell cytoskeleton in cell migration and would like to know whether there is any difference if using fluorescent labeled mAbs (actin and tubulin) or small molecular specific binders (paclitaxel and phalloidin) and which one gives a better signal to noise ratio? Thanks.
Hi,
it depends if you want to perform live-cell imaging or not. For live-cell you will need something like SiR-tubulin or SiR-actin or plasmids. But most of the live-cell dyes somehow interfere with dynamics of the cytoskeleton. SiR-tubulin is based on Docetaxel and stabilizes microtubules. If you want to do immunolabelings you might have to play around with fixation protocols. Methanol-fixation or PFA with 0.01% glutaraldehyde in cytoskeleton buffer usually lead to good results. In fixed cells you loose the dynamics but you don't have the side-effects of the drugs.
Best,
Andreas
Phalloidin works much better than actin antibodies for fixed cell labeling. For microtubules, there is a monoclonal antibody (clone DM1A) that works great.
Hi Jrhau,
For live-cell imaging, F-actin binding peptides/proteins genetically labelled with FPs (e.g. EGFP-LifeAct, Uthropin) perturb much less actin dynamics than F-actin binging small molecules coupled to fluorophores (e.g. Sir-actin). You may want to go for the latter option when cells cannot be transfected or infected. However, Sir-actin is a derivative of pahlloidin and, in our hands, should be diluted far more than recommended to avoid stabilisation of actin filaments.
For fixed specimens, I would recommend having a look at our recent publications:
Article PFA fixation enables artifact-free super-resolution imaging ...
Article ARTICLE Flat clathrin lattices are dynamic actin-controlled ...
and associated protocol:
https://www.nature.com/protocolexchange/protocols/4721
Best regards and good luck,
Metello
There are basic differences between antibody and others that you mention. First of all, paclitaxel is not recommended for MTs as they will interfere with microtubule dynamics and will only bind to certain MTs not all of them. The best for MTs is the Monoclonal antibody clone TUB2.1 labelled with Cy3. You can also do unlabelled antibody and use a secondary antibody labelled with Alexa488 or Cy2.
For filamentous-Actin (F-actin) of course labelled phalloidin is a very good choice. A very good combination is Alexa 488 phalloidin + Cy3 labelled tubulin antibody. Remember you have to label with Phallodin before you put the blocking agent otherwise it won't work. Phalloidin also does not work very well with paraffin sections.
Phalloidin only stains F-actin, if you want to stain G-actin as well F-actin then you should use an antibody against actin labelled with Cy2 or Cy3. This will give you a bit more background because of the G-actin, but this should be helpful for accessing G-actin levels in the cell.
Overall, I like the following combination: Alexa 488 phalloidin, Cy3 labelled tubulin antibody and DAPI for nuclear stain.
Hope this helps!
Regards
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