[Zbrafish] Re: whole mount in situ hybridization problem

Cheuk-Chung Tony Ho wind.4ever at gmail.com
Tue Aug 1 21:21:21 EST 2006


As far as my experience can tell, I would definitely check out the
following points.

1. Probe
This may sonds trivial, but i will make sure that the probe binds your
specific target transcript. I have had experience with probe around 1kb
in size and they do pretty good. Therefore, I don't think it's a size
problem.

2. Wash Condition
I use a gradient. I wash with 2x SSC (2x15minutes), and then follow by
0.2x SSC (2x30minutes). The whole wash proceedure is at 65C.
I think this Post-Hybridization Wash step is critical to getting a
clean background. However, since your positve control shows a clean
background... it would be hard to make a judgement.

3. Embryo
Sometimes, it's just one bad batch of embryo. Have you tried with a
fresh batch?

Hope this helps.
Keep us posted for any update.

Yours,
Tony

SmallPotato wrote:
> 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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