IF fixation - (Nov/14/2005 )
I have been trying to probe chondrocytes expressing the E7 protein (15 kDa) in an IF assay using a mouse anti-E7 antibody (neomarkers) in conjunction with a goat anti-mouse alexa 488 conjugated secondary ab. (1:100 dilutions). I know my secondary is good and i have used 2 different lot numbers of the primary antibody....so i seriously doubt that the antidodies are the problem.
Sadly however while using the correct filter i get NO fluorescence from these cells following a 30- 60 min fixation in 100% methanol. Having reverified everythng else i am concerned that the problem may be my fixation step. Is it possible to lose such a small protein from the cells in that time?
btw...i block and probe in 5% skim milk in PBS and i probe in the same.
Should i consider alternate forms of fixation? formalin perhaps?
first make sure that your primary is IF grade. Ask neomarkers if you can use them. Sometimes some antibodies dont do too well if used for IF. The batch might not be the problem...but it might be the type of antibody.
I am not sure though if the fixation is an issue. If u want to try it our anyway...go with 4% paraformaldehyde for 20 mins and then 0.1% trition for 5 minsx 3.
go on with 2h primary and 1h secondary at RT.
Can you give more details of your procedure so I can help? Upon first glance I see you do a 30-60 minute fixation in methanol. This is WAAAAAAY too much. Try 5 minutes in acetone, methanol etc or 20 minutes in 2% PFA/PBS followed by permeablization as Pria suggested. I find many antigens are ruined by methanol so I prefer acetone or formalin depending on my antigen of interest.
we use PFA/PBS (4%) for only one minute and the keratinocytes are sufficiently fixed for IF; do chondrocytes require much more stringent fixing methods??