Dear fellow researchers:
Ive been doing this experiments for a while but recently I've observed that not all of my cells are getting stained with FITC-Phalloidin. Can anyone suggest possible scenarios of this? Here's my protocol:
MCF-7 cells
4%PFA- 10 minutes
0.2% TX-100 - 10 minutes
2% BSA blocking - 1 hour
FICT Phalloidin incubation - 20 minutes
IMAGES (40X) ATTACHED FOR MORE CLARIFICATION
One can see a lot of cells with DAPi staining but only a few with FITC-Phalloidin.
Hello Dhawan!
I think there may be the following reasons for that:
1. I can see the effects of photobleaching in FITC-Phalloidin channels may be one of the reason as FITC is the most vulnerable fluorophore for photobleaching and DAPI is much more stable.
2. You can also try decreasing the blocking time (reduce it), as it usually is instrumental in case of immunostaining but I am not sure how it acts on the staining in dyes like Phalloidin stains.
3. Its better to merge your images through softwares like ImageJ, Adobe Photoshop or the software provided with your fluorescent microscope. This will better justify whether the actin-stained cells are stained for DAPI or not, because usually it doesn't happens.
Best wishes
Ali
Hi Syed!
Thanks for the suggestion. The samples are mounted using Prolong Diamond antifade. The samples are observed immediately after preparation with little to no exposure to light. My past experience with Phalloidin-FITC has been that it stays stable for a while. So I can confidently rule out that its due to photobleaching.
Reducing blocking might work though. Worth a try, I think.
I did merge them using the microscope's software and only 3 cells stained for Phalloidin + DAPi. All other stained for DAPI alone. Unfortunately I don't have the image the moment.
I am also considering the case that our Paraformaldehyde might be expired so it may be due to improper fixation.
Thanks again man!
Another possibility may be to fine tune the permeabilization step, because if the cells are not completely permeable to the dye the dye might not enter the cells to stain them. Though 0.2% Triton for 10 minutes is fine but it can be one of the way to fine tune your stain.
Good Luck!
Hi Udesh Dhawan ,
Did you find a solution to this? I'm having the same problem, all my cells (HUVECs) are stained with Hoechst, but I'm seeing very patchy rhodamine phalloidin staining. Some cells are stained brilliantly, some are dim and some are not stained at all. Seems to be fairly random!
Protocol:
- 15 mins 4% PFA
- 5 mins 0.5% Triton (a colleague of mine misses this step out completely with no problems)
- 1 hour phalloidin/Hoechst staining in 1% BSA/PBS
Any suggestions are welcome!
Hi Emma,
Try seeding cells at a low density and observe if you see differently stained cells. I realized that in my case, cells were at a very high density, crowded, causing not them to be stained evenly. In my case, I switched to Ready probes 488 actin green reagent. It worked very nicely. Try this. I have also observed that the way you pour your chemical the coverslip is also very important. If some regions have a higher amount of the phallodin, they will be stained brighter And area where the amount isn’t enough, will be stained less brightly. How do you pour the chemical on the cover slip?
Hiii,
Thanks for getting back to me!
My cells are already at a pretty low density (I'm looking at cell spreading in 1 hour time points), I'm just doing the staining in a TC plate, 24 well, 0.5ml liquid per well. Sometimes it works perfectly and all the cells are stained perfectly, other times it doesn't work. I'm not really sure what causes the partial staining.
I've attached a picture so you can see what I mean!
Yeah I had identical issues. Is It possible for you to try Ready probes 488 Actin green? It's quite inexpensive and relatively simple to use. Just add 2 drops/ml of PBS or medium.
One hour of staining is a relatively long time. Have you tried the recommended 20 minutes? It may be possible that after you add chemical to the wells, after a while it is not evenly distributed. Perhaps you can try keeping it on an orbital shaker at a low rpm so it comes in contact with all cells through the incubation period.
Yeah, I did try 20 minutes first, and then when this didn't work I tried to re-stain the same samples several times but nothing changes, I even tried a shaker, and higher concentrations of phalloidin... It's very frustrating!
Anyway, thanks for all your help, I'll look into the Ready probes Actin green :)
You're welcome! Although, Phalloidin is supposed to display affinity binding to F-actin filaments. However, what if there are no F-Actin filaments? This is just a wild guess. I am not sure if unhealthy cells display decreased F-Actin. In that case, even if everything else is right, there is just no place for Phalloidin to bind, resulting in no fluorescence. Perhaps doing a literature review on this might help? It is a possibility if both chemicals, Phalloidin and 488 ready probes give similar results.
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