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

[Snehalata Kadam]

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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> Today's Topics:
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>    1. biotin-avidin-HRP background problem in doing FISH (Ying Wen)
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> ----------------------------------------------------------------------
>
> 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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>
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