[Zbrafish] Re: whole mount in situ hybridization problem

SmallPotato jxie at unity.ncsu.edu
Thu Jul 27 11:46:09 EST 2006


Yes, I used DIG-labelled probe. AP conjugated secondary antibody. It
took 2 days at RT for BM purple sutstract to give me some colour. But
both sense and antisense give me colour. But my blank control(without
probe) is completely white. I tried NTB/BCIP. This substract gave me
colour immediately. But both sense and antisense are the same.

My hybridization Tm is about 67 degree. I use 2xSSC and MBT solution to
wash. The most important imformation from my experiments is, I have a
positive control probe from other lab(they gave me RNA probe only). In
my protocool, the expression pattern of this positive control gene is
beautiful. So I don't think my protocol have any problem. I am an
experienced worker regarding ISH, since my in situ results in Xenopus
embryos is beautiful.

I am frustracted with changing a lot of conditions: decrease
hybridization Tm to 60 degree, use full length to hybridize, fragment
the full length to 50-150bp to hybridize. But the result were not good
at all. I don't know where my gene is expressed!!!

The only thing I am very worried is: if my sense probe of my gene will
bind to the mRNA. That is troubling. Is there any way to figure this
out?

Thanks,
Small Potato.


Cheuk-Chung Tony Ho wrote:
> For starters, what protocol did you followed? and what is the
> hybridization and wash condition? I racken you use DIG-labelled probe,
> mate?
>
> Tony
>
>
>
> SmallPotato wrote:
> > Hi,
> > I have cloned my gene using RT-PCR. Now I want to see its expression
> > pattern using whole mount ISH. My postive control using the other probe
> > works. But my gene have trouble in getting signals.I have tried 1.1kb
> > and 2.0kb probe. None of them works.Do you have any suggestions?
> > Thanks,



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