VEGFR:VEGF using antibody Gv39M

[enaik]

Hi everyone,

I've been having no luck staining with this antibody (only in a two step protocol but this has apparently worked for some published work that doesn't provide a detailed protocol). I've tried it on frozen sections, air dried for 1h, fixed in -20 acetone for 10min and then air dried for 30min at 37C. These sections are then incubated with biotinylated primary at 1/20 dilution overnight at 4C and then processed by standard DAB methods. My positive control was E13.5 and E14.5 embryos, which did not have any staining either.

Any help would be greatly appreciated!!!!

Edwina

[excalibur]

What is your DAB procedure? You say it is a 2 step protocol.
After the primary, you should have a biotinylated secondary, then an HRP label, and then the DAB.

Also dry at room temp after the acetone instead of 37C.

[enaik]

Thanks for your reply,

I use a biotinylated primary, then HRP and DAB. I suspect this isn't sensitive enough but saw this published as a protocol for frozen sections. I think I will look into purchasing the unconjugated form and then do a 3 step procedure.

Thanks again.

Edwina

[ImmunoNYC]

I don't have much to add except to point out that VEGF is notoriously difficult to stain for. If I get a chance I will look into this antibody and get back to you.[/color]

[neusci]

Have you tried using something other than acetone to fix?  I had to use 2% paraform on one particular protein because the acetone was too harsh.  Maybe methanol for you?  We used to stain for VEGF-R and VEGF on paraffin cuts without too much trouble.  The protocol for that was the usual.  Didn't even need AR.