[Histonet] Re: Rabbit Anti-sera Background

[Johnson, Teri]

Trey,

Perhaps your detection system is TOO sensitive for this antibody.  In
some cases, rather than take the dilution of the primary antibody out to
a ridiculous level, we have opted to use an HRP labeled anti-rabbit
secondary if we find the positive staining very intense and background
too high.  

Also, have you tried different antigen unmasking procedures?  It could
be you don't actually need the power of EDTA to open up the binding
sites.  In any antibody development, it's best to try an enzyme
(trypsin, pronase, protease) digestion in addition to a citrate buffer
type formulation, and also keep one set of slides with no pretreatment.
If your results are weak or negative, then move on to the more powerful
enzymes and high and/or low pH HIER.

It could be you're hitting it with much more power than it really needs
to stain well.

I do really admire your approach to troubleshooting.  Well done.

Hope this helps,

Teri Johnson
Managing Director Histology Facility
Stowers Institute for Medical Research
1000 E. 50th St.
Kansas City, MO 64133