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


1) A number of factors could cause the actual abundance of this mRNA in your
tissues to be lower in the mouse, under your conditions, even if the probe
works comparably well across species (and typically the sequence is pretty
close between rat & mouse), e.g. age, stress, experience.
2) Fixation should improve the anatomical resolution of your signal, but I
wouldn't think less fixation would reduce the signal.  Some protocols call
for eliminating fixation altogether.  But high concentration of fixative
will definitely interfere with the penetration of probe into the tissue,
especially for ribo's.  So I suspect that your colleague is on target.  Of
course the proof will be empirical comparison!  Are you using PFA/PBS?
     Good luck!   (BTW, I'm an alum of the UM biopsych program [back in the
dark ages when we used to be called psychobio]--please convey my regards!)
Susan Bachus, Psychology Dept., George Mason University
----- Original Message ----- 
Sent: Friday, April 16, 2004 2:53 PM
Subject: [Histonet] mouse vs. rat in situ; overfixed vs. poorfixed

> Dear All,
> Recently I have tried radioactive in situ hybridization on mouse brain
> 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
> similar intensity, I need to lay down the film on mouse tissue longer time
> twice much as on rat tissue.) I am not sure if it is normal for mouse
> get lighter signal or the rat in situ protocol just is not optimal for
> 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
> 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
> 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
> fixing time. By the way, the PFA was stored in a caped container in
> Well, same fact but totally different viewpoints. Any comment?
> THanks.
> Pengwei Yang
> Biopsychology Program
> University of Michigan
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