cryosection - blocking (Nov/02/2009 )

hello

I work eith cryosection and myc antibodies.
untill now I worked with tisuue culters with the same antibody.
and I used 5% milk for blocking.

Does blocking with milk is good for my cryosection as well?

thanks,
Shani

Depends what tissue you use, is it Human tissues ? Milk is the cheapest blocking agent other possibilities are BSA, Gelatin or sometimes animal sera matching the host where the first antibody has been raised

pesji on Nov 2 2009, 03:59 AM said:

I'm using rat`s heart.
I try yo use milk, and I got a alot of backroung.
in the control (only second antybody, without the first) there wasnt staining at all, so I think that maybe the problem was with the blocking method.

is that sounds reasonable?

What sort of detection system are you using? I would reccommend BSA with biotinylated antibodies or biotin-streptavidin detection because milk has a lot of biotin. Another thought....if using HRP or AlkPhos secondaries, you need to quench the endogenous peroxidase/phosphatase activities in the tissue before applying your secondary.

jah on Nov 2 2009, 05:43 AM said:

I prefer the flouracent method, so I use cy3 antibody as secondary.