Fixing cells for performing immunoflourescense? - Help with protocol (Aug/05/2005 )

Hello! I hope you can help me better my protocol for immunocytochemistry.
I want to make flourescent labeling of my vascular smooth muscle cell cultures.
I have tried it before where I used 5% H2O2 in ethanol for fixing the cells onto the small glass slides.
But the cells wouldnt stick during the procedure.
Do any of you have a tip?

I hope you can help me,

Thank you for a great forum.

Karina

I would fix in 1-4% PFA in PBS for 15 minutes at RT.
Generally H2O2 is not considered a fixative in the practical sense and is normally used for blocking when doing IHC instead of fluorescence based protocols.

Thank you! I will try it today.

Hi,
PFA fixation is working quite well. However, if you want to detect an intracellular protein you have to permeabilise the cell before staining (0,5 % Triton X-100 in PBS) because PFA just crosslink all protein. A very fast and easy method is to simply fix the cells in ice cold acetone (keep it @ -20°C). Acetone precipitates all proteins and washed away all lipids. Therefore no permeabilisation is necessary. However, some membranous proteins might get lost. The advantage is: you can keep the fixed cells in acetone for a long time.
Good luck – CCS