[Drosophila] Re: Dros Digest, Vol 8, Issue 5

Ying Wen wenvt5 at yahoo.com
Fri Dec 30 11:51:01 EST 2005


Dear Snehalata,

Thanks for your reply. We tried regular FISH and the
signal is too weak (we used Molecular Probe as same as
you). That's why we went to TSA kit. I think our
probes are good since our regular AP staining works
beautifully by using the same probes. So there must be
some techinical trick out there that we are missing. I
am particularly wondering if you do preabsorbtion of
the first and secondary antibodies. Another question
is what kind of blocking reagent you are using. Thanks
a lot.
We got the TSA kit from Molecular Probe, the catlog
number is T20915 and T20932 (they offer many TSA kits
with different dyes and species).  There is a document
coming with the kit, but we also use some information
provided in Kosman's protocol (online supporting
material of Science paper, 2002).  Let me know if you
have any question.

Ying


--- Snehalata Kadam <snehalvk at caltech.edu> wrote:

> Dear Ying
> I am a Post Doc at Caltech...........
> well what we do normally do for  the dig and  bio
> probes we use  
> anti-dig, anti-bio from Roche 1.400 dil. For fitc
> and  dnp probes we  
> use anti-fluorescein, anti-dnps from Molecular
> probes and all the Alexa  
> fluors 488, 555, 647 1:400 from Molecular probes as
> the  
> secondary´s............
> everything works very well as long as the probes are
>  
> good...............I never needed TSA amplification
> for FISH.
> the protocol is from:
> www.biology.ucsd.edu/~davek/treatment.html
> 
> By the way,
> I have a question for you about TSA amplification. I
> have once for the  
> MAPK Ab, it worked well once but second time it did
> not work. Could you  
> please let me know the protocol used for TSA
> amplification and the  
> company from where you purchase the kit?
> Thanks a  lot
> Snehalata
> 
> On Dec 18, 2005, at 9:00 AM,
> dros-request at oat.bio.indiana.edu wrote:
> 
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> >    1. biotin-avidin-HRP background problem in
> doing FISH (Ying Wen)
> >
> >
> >
>
----------------------------------------------------------------------
> >
> > Message: 1
> > Date: Sat, 17 Dec 2005 06:35:42 -0800 (PST)
> > From: Ying Wen <wenvt5 at yahoo.com>
> > Subject: [Drosophila] biotin-avidin-HRP background
> problem in doing
> > 	FISH
> > To: dros at magpie.bio.indiana.edu
> > Message-ID:
> <20051217143542.40156.qmail at web53512.mail.yahoo.com>
> > Content-Type: text/plain; charset="iso-8859-1"
> >
> >     Dear everone,
> >
> >   I am a research asssitant working in Cincinnati
> Children's Hospital  
> > Research Foundation. I am currently doing
> dual-label FISH (Fluorecent  
> > Insitu Hybridization). I choosed the following
> schema:
> >
> >   1.  dig labeld mRNA probe of gene1---------mouse
>  
> > anti-dig(Roche)---------Goat anti mouse with
> HRP(Molecular  
> > Biology)------Tyramide Signal Amplification
> (Molecular Biology)
> >
> >   2. biotin labeld mRNA probe of
> gene2------------strepavidin-HRP  
> > (Jacson Immuno)------Tyramide Signal Amplification
> (Molecular Biology)
> >
> >   The first one (dig) gave me nice staining, but
> the second one  
> > (strepavidin) always gave high background. I know
> the biotin labeled  
> > probe is good, since when I use this probe tested
> with mouse-anti-bio  
> > antibody, then followed by Goat-anti-mouse-HRP
> second antibody, it  
> > works beautifully. So, the problem could be
> biotin-avidin problem.
> >
> >   Did anyone experience same problem when doing
> FISH? Appreciate it!
> >
> >   Sincerely Yours,
> >   Ying Wen
> >
> >
> >
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> > End of Dros Digest, Vol 8, Issue 5
> > **********************************
> >
> Snehalata Kadam Ph.D
> Division of Biology 114-96
> CALTECH - Broad Center
> 1200 E. California Boulevard
> Pasadena, CA 91125
> email: snehalvk at caltech.edu
> Lab: 626-395-5951/5952
> Home: 626-584-0886
> Mobile: 626-354-5473
> 
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