5-HT2A not working! Help!!

[camille]

Hi,
I've got a big problem with my monoclonal mouse anti-5-HT2aR antibody. i'm working on free floating rat and mice sections (40microm thick) just perfused animal, not paraffin embedded.
there is very little staining with DAB, no specific staining at all and the controls show very little background (which is possibly the only positive thing about it!!)
I thought it was the primary antibody that had gone off but I tried a new one and still nothing. the secondary is the right one and new. my supervisor had tried it a few months ago and it worked OK.
I really can't think of what I'm doing wrong please help!! :cry:

camille

[richard03]

Did you incubate the primary long enough? What is the concentration of your primary?

[camille]

the concentration I use is 1:500 which is double what was used before when it worked and I left it incubating for 48hrs.
Could temperature be a factor, maybe rather than RT incubate at 37 degrees C for the same or less time?

[richard03]

Higher temperature will help. Also try to increase conc to 1:200.

the concentration I use is 1:500 which is double what was used before when it worked and I left it incubating for 48hrs.
Could temperature be a factor, maybe rather than RT incubate at 37 degrees C for the same or less time?