Re: [Histonet] In situ hybridization

From:Jimmy Lao

Hi, Carlene Zindi,
It seems you are going to do In situ hbridization on
slides. I would like to answer your question based on
this point with my experience.
1. We used dig conjugated RNA probes like fgf8, otx2,
Shh and so on for mouse embryo and the brain, limbs
from bigger than E12.5 mouse.  They work fine.
2. The fix method is that 4% paraformaldehyde in PBS
at 4 C overnight with gentl nutation. Wash in PBS 5
min at room temperature for two times. embed in OCT
crystat section 10-14um thickness.
3. About 1Kb anti-sense probe is reasonable, and it is
better using 3' UTR(untranslation region) due to not
much conserved.
Hope this help.
Zhimin Lao

--- "Carlene L. Zindl" 
> Hello,
> Does anyone have experience with non-radioactive in
> situ hybridization of
> frozen mouse tissue??  If so, I would appreciate
> advice on details such
> as: 1) is biotin or dig better? 2) what is the best
> way to fix this
> tissue?  3) what is the optimal size RNA probe?
> Thank you for any pertinent information.
> Sincerely,
> Carlene Zindl
> University of Alabama at Birmingham
> -OR-
> _______________________________________________
> Histonet mailing list

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