Currently i have been using 90% ice cold methanol and incubating either on ice for 30 minutes or at -80 degrees for a few days. I need to permeabilise my cells for an intracellular phospho staining and so far I'm not getting a very good signal at all. I know there a multiple methods for permeabilisation and i just want to know some other methods i can try that someone has personally andused had success with.
Thank you in advance!
To add to Hemavathi,
I fix in 2% PFA for 20 min, wash with PBS and then perm with 0.2% saponin over night at 4C. The longer longer the perm the better, especially if you are looking at nuclear proteins. The next day I wash with PBS, then add my antibody cocktail resuspended in saponin and stain for at least 30 min on ice. It is a longer protocol but works beautifully.
IMPORTANT: Some antibody clones will work better with methanol fixation and some will work better with PFA fixation. If you have tried different perm protocols, I highly recommend trying a different clone.
Hello Kyal. How about Triton X-100 for 5 minutes? The article here states how they bind antibodies to both the adherent (extracellular) and intracellular bacteria, and they permeabilise the cells with Triton X-100.
Article Thrombospondin-1 promotes cellular adherence of Gram-positiv...
Hi Kyal,
In our lab we tried using methanol but we also did't get good results. So, we started to use saponin. Briefly, 0.2% saponin ( ~1 million cells suspend in 100 microl of sap) leave it for 5min and then add antibody and incubate for 45min in ice.
Good luck
Hi Kyal,
Do you want to do intracellular staining for flow cytometry?
We use PFA for fixation followed by permeablization with PBS/0.1%Tween-20.
Alternatively, there are some good Fix/Perm kits available from companies (we use the ADG Fix and Perm Kit).
Best
To add to Hemavathi,
I fix in 2% PFA for 20 min, wash with PBS and then perm with 0.2% saponin over night at 4C. The longer longer the perm the better, especially if you are looking at nuclear proteins. The next day I wash with PBS, then add my antibody cocktail resuspended in saponin and stain for at least 30 min on ice. It is a longer protocol but works beautifully.
IMPORTANT: Some antibody clones will work better with methanol fixation and some will work better with PFA fixation. If you have tried different perm protocols, I highly recommend trying a different clone.
Thank you all for your replies.
I am going to give the Triton, Tween and definitely Saponin a try!
For intracellular protein detection I would fix cells in 1-4% paraformaldehyde for 10 mins and then permeabilise overnight in buffer containing 0.2% w/v saponin at +4C. You can get away with permeabilisation for 30mins at 37C but you may get higher background labelling.
I have just come across a link to a paper that may be of interest: http://www.ncbi.nlm.nih.gov/pubmed/14505311
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