[Histonet] Thanks! (about few questions about IHC DAB staining)

[yangpw <@t> umich.edu]

HI, All,

Thanks so much for the warm replies I got. They are very helpful. About the
light staining with DAB, I have some more details I would like to supplement. 

1)The DAB I used is from Sigma (DAB tablet D5905, 10 mg/tablet, store at -20 c).
I made fresh DAB solution about one hour before I used it (10 mg DAB, 10ul 30%
H2O2 in 30ml TBS, I add DAB first and stir until resolve and filter it using
0.2 um filter, add H2O2 right before using it). The interesting thing is after
1.5 hours incubation in DAB solu, the background did not enhanced much and
actually was relatively low. I will try ordering a new batch of DAB and H2O2. 
At the same time, do I need to increse the concentration of DAB or make other
changes to my DAB protocol? There are also some commercial liquid DAB
available, are they reliable after long-term storage?

2) The stains I got are doubtless c-Fos staining and I store aliquoted primary
antibody in the -20 c freezer. So there is no repeat thaw/freeze problem and
the same batch of primary antoibody was good in previous staining. THe antibody
came in with 200 ul and i just simply divided it into several 10 uls and stored
at -20c. I notice some labs use glycerol to dilute the antibogy before put in
the freezer. Does that help to sustain the reactivity of antibody?  

3) The Vector ABC kit I used is still before expiring date. The concentration of
ABC reagent I used though was 1:1,000 of both A and B reagent instead of what
the company recommend. And I have tried this kind of concentration many times
before and it worked fine. But I am not sure if I need to increase the
concentraion since the ABC kit I used has been in refrig for several months. 

Any more suggestion is appreciated. Thanks.

Pengwei Yang 

Biopsychology Program
Psychology Depart.
University of Michigan