[Histonet] few questions about IHC DAB staining


HI, All,

I have been doing c-Fos IHC stainging and using Vector ABC kit and DAB as
chromagen. THe protocol I use worked fine in the past, but recently I have
confronted couple problems. ANy suggestion would be greatly appreciated!!  

1) The staining is kind of light. I have incubated tissues (rat brain, 30 um,
cryostat-sliced, 4%PFA perfused and 30% sucrose cryopretected before slicing,
free floating) in DAB color reaction solu for one and a half hours, and the
cell staining is ok under microscope but still light comparing to the intensity
I used to get. Background is ok. In the past I used same protocol, same
concentration of antibody, and only need eight minutes to reach pretty decent

2) The sections tend to stick to each other during the incubation of primary
antibody (at 4c for 48 hours), which cause uneven staining. The overlaped
tissue tends to be light stained. I use specific staining plate, which has mesh
under the wells to facilitate the tissue transfering during washings. Is it
possible related to the staining plate, or to shaking speed? How can I avoid

3) Recently I am going to try calbindin (D-28k) staining on rat brain to
delineate the core and shell of nucleus accumbens. I have searched some papers
and it seems many people use monoclonal mouse anti-calbindin (Sigma). But I am
kind of concerned about the nonspecific crossreactivity between secondary
antibody and rat tissue, since rat and mouse are close. Some people use
polyclonal rabbit anti calbindin, but it will crossreact with calrentinin,
which might compromise the clear delineation between core and shell. Any
opinion is very welcome.

Thanks in advance. 

Pengwei Yang

Biopsychology Program
Psychology Depart.
University of Michigan

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