Hi,
Iam trying to study CDT1 and Gemenin, for G1 and G2 phase of cell cycle. Iam not able to get clear signals in the nucleus by immunofluorescence. Iam fixing my cells with 4% PFA. This this good or methanol fixation good for nuclear protein.
Thank you
Dear Jaya,
PFA fixation will not be enough to permeabilize the nuclear membrane, so you will have to add an extra step with TritonX, Tween20, Saponin or similar.
If you choose to use methanol or 100% ice-cold acetone you will both fix&per, so there is no need for extra steps.
Good luck!
Matteo
4% formaldehyde PBS is fine. Fix for about 10 minutes. Then wash twice with PBS. Your problem seems to be with permeabilization. Make sure to include a stronger detergent (either np40 or triton) at a concentration of 0.3-0.6% during a blocking step that should last 30 mins or so prior to your incubations with primary. This will probably be enough for most intracellular proteins, but you can include a lower concentration of strong detergent .15-.2% (of np40 or triton) or use .05-.1% of weaker detergent tween20 in with your primary and secondary to make sure your antibodies permeate. I recommend a strong detergent of 0.2% for nuclear proteins.
Hope this helps,
Bill
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