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


Dear Dr. Yang,
I am not familiar with differences of ISH on mouse and rat tissues. 
However, on old vs fresh paraformaldehyde, the old one has more power of 
cross link, ie more likely to cause over-fixation. Because, upon storage, 
PFD condenses to poly PFD, a chemical reaction occur easily at neutral pH 
at 4oC.
Best regards,
James Guo
At 02:53 PM 4/16/2004 -0400, you wrote:
>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 
>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 
>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?
>Pengwei Yang
>Biopsychology Program
>University of Michigan
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