Methanol oor PFA to fix cells for nuclear protien analysis by immunofluorescence - (Mar/17/2015 )

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

It's not usually about the location of the protein, it is the protein itself and the antibody used to detect it.

Make sure your antibody is capable of detecting the native protein (i.e. won't just detect the denatured form). You may need to test a range of different fixation methods and or antibody incubation buffers.

Also make sure you are permeablizing the cell properly.

The data sheet shows that it can be used for IF and they also have a beautiful fig showing the fluorescence in the nucleus. Thats why I went with that company, but some how Iam not getting it.

Are you following the procedural steps as is recommended by the company? Do they specify how they obtained such a nuclear protein staining as the one in the photo?

no they did not give the protocol. when I asked them, they sent me the general procedue, that I find in the website