[Histonet] mouse vs. rat in situ; overfixed vs. poorfixed

[yangpw <@t> umich.edu]

Dear All,

Recently I have tried radioactive in situ hybridization on mouse brain tissue
with mouse-cfos riboprobe. I found that with same in situ protocol, the signal 
of mouse is lighter than the signal of rat-cfos on rat tissue (to reach the
similar intensity, I need to lay down the film on mouse tissue longer time as
twice much as on rat tissue.) I am not sure if it is normal for mouse tissue
get lighter signal or the rat in situ protocol just is not optimal for mouse.
Any suggestion? If I need to modify my rat protocol for mouse, what aspect
should I change?    

The second question is kind of interesting. Still about in situ, I tried two in
situ on mouse brain tissue (the tissues are two copies from same animals, and I
use same in situ protocol). The staining solutions are all the same except the
4% PFA I used to fix the tissue. In the first staining I used a three-month old
PFA, and in the second staining I made fresh PFA. The signal of second staining
is still kind of light but much more intense than the signal from first
staining. My conclusion is in the first staining the tissue was poor fixed
because stale PFA was degraded. So to improve the staining, I will try fresh
PFA with longer fixing time. But one of my collegue has a exactly contrary
opinion. He thinks in the first staining, the tissue was over fixed becuase the
old PFA became more concentrated. So he suggest using fresh PFA and shorter
fixing time. By the way, the PFA was stored in a caped container in fridge.
Well, same fact but totally different viewpoints. Any comment?

THanks.

Pengwei Yang

Biopsychology Program
University of Michigan