[Zbrafish] Re: whole mount ISH

finchg from ohsu.edu via zbrafish%40net.bio.net (by finchg from ohsu.edu)
Wed Jul 9 17:04:45 EST 2008


When you say none of them are working do you mean no signal or embryo-
wide signal?  Also, are you sure your gene of interest is expressed at
the 5dpf timepoint as a lack of signal may not necessarily indicate a
defective protocol?

Having tried several protocols for zebrafish in situ and read through
nearly every one I could find on the internet, I would recommend the
Thisse et al. protocol available from the Moens Lab website: <http://
www.fhcrc.org/science/labs/moens/Protocols/>.  This protocol has
worked consistantly even at 5dpf timepoint.

For 5d embryos, use of PTU from ~12 hrs. to prevent pigment formation
is superior to later (potentially destructive) post-fix bleaching.  In
preparing the fish for hybridization, protienase K digestion time may
need to be optimized due to product variability.  I use Roche PCR
grade ProK at 10ug/ml and would recommend a digest time of at least 30
min for 5dpf.  Overdigestion is apparent when the embryos break apart,
underdigestion may be less evident, but will effect staining of deep
tissues.  If you are concerned only with superficial structures than
fine tuning this may be less relevant.  I also have much more
consistant results mixing my own staining buffer using Roche NBT/BCIP
(as in the recommended protocol) as opposed to the Roche hybridization
kit which uses "BM Purple AP substrate".

If you have access to a probe that you know works, try it at the same
time as any untested/troublesome probes as a positive control.  This
way you can quickly determine if problems are with probe preparation
or with some other aspect of the chemistry.  The same could be said
for trying another earlier "easier" time-point at the same time.

-Gabe Finch
Nicolson Lab
Portland, Oregon

On Jul 8, 6:59 am, Els <els.janss... from gmail.com> wrote:
> Hi,
> Does anyone ever done a whole mount ISH on 5dpf embryos that worked?
> I already tried practically every protocol but none of them are
> working.
> Greetz,
> Els Janssens
> Els Janssens
> PhD-student
> Animal Physiology and Neurobiology Department
> Neural Circuit Development and Regeneration
> Naamsestraat 61, bus 2464
> B-3000 Leuven
> Belgium
> Phone:  +32 16 323987
> Fax: +32 16 324262
> E-mail: els.janss... from bio.kuleuven.be

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