Cell fix that doesn't permeabilize

[Caroline Bass]

Hey Guys,

I thought the folks on this forum might be able to help with this issue. I’m doing in-cell western blots using the Licor system. You can look at cell surface protein if you don’t permeabilize the cells, and then total if you do permeabilize the cell. The problem is that we used a standard formalin fix and it seems to have been a little rough, we suspect some of the antibody is getting into the cells after fixation. Can anyone recommend a gentle fix that does not permeabilize cells? FYI, we are using N18 and SHS-5Y5 cells in a 96 well format. My only thought is to use a fix that I have for primary neurons, it seems a bit gentler, but I really don’t know if it’s permeable or not.

Thanks!

Caroline Bass

[F Javier Díez Guerra]

Hi Caroline,

in our hands, aldehyde fixation makes cells leaky to different extent depending on cell type and fixative. If you want to label proteins exposed on the cellular surface, you will prefer to wash and cool down your (adherent) cells to 4-8ºC and then incubate with your primary antibody (at least 60 min). After thorough wash, then you can fix and permeabilize to label intracellular antigens.

Detailed protocols could be easily found if you look for methods for "in situ" quantification of endocytosis.

Best wishes


El 10/03/2010 18:37, Caroline Bass escribió:

Hey Guys,

I thought the folks on this forum might be able to help with this issue. I’m doing in-cell western blots using the Licor system. You can look at cell surface protein if you don’t permeabilize the cells, and then total if you do permeabilize the cell. The problem is that we used a standard formalin fix and it seems to have been a little rough, we suspect some of the antibody is getting into the cells after fixation. Can anyone recommend a gentle fix that does not permeabilize cells? FYI, we are using N18 and SHS-5Y5 cells in a 96 well format. My only thought is to use a fix that I have for primary neurons, it seems a bit gentler, but I really don’t know if it’s permeable or not.

Thanks!

Caroline Bass