secondary antibody from immunostainigs

[anh2006]

Yes goat serum not rabbit serum, exactly.

If the problem is indeed high non specific signal I would recommend blocking with PBS, BSA 1%, Goat serum 5 to 10% if your secondary antibody is made in goat. I routinely use highly cross adsorbed goat anti rabbit coupled to alexa fluor (Invitrogen) diluted 1:200 for immunostaining mouse tissue and get no NS signal.

If binding by Fc receptor can be a problem (I know nothing about receptors in platelets membrane), you can use Fab'2 fragment of the above secondary antibodies.

Hope this can help

bruno

 

Bruno SAUBAMEA

 

EA 3621 & Service Commun d'Imagerie Cellulaire et Moléculaire

 

Faculté des Sciences Pharmaceutiques et Biologiques
Université Paris Descartes
4, avenue de l'Observatoire
75006 PARIS

 

tel : [phone]
fax : [phone]

----- Original Message -----

From: [hidden email]

To: [hidden email]

Sent: Tuesday, March 10, 2009 10:32 AM

Subject: Re: secondary antibody from immunostainigs

hello Bruno SAUBAMEA
what i could see from my stainings, it seems that the background is so 
high that the specific signal canot be seen. to get rid of the high 
background i tried washing with high salt PBS but it seems to have 
done no improvement to my samples.
  thank you
  Shuchi Gupta
Rudolf Virchow Centre for experimental medicine.
Uni Wuerzburg
Germany




Quoting Bruno Saubaméa <[hidden email]>:

> Dear Shuchu,
> what do you think is the problem :the bg is low but you don't have a 
> specific signal or the bg is so high that the specific signal can't 
> be seen?
> bruno
>
> Bruno SAUBAMEA
>
> EA 3621 & Service Commun d'Imagerie Cellulaire et Moléculaire
>
> Faculté des Sciences Pharmaceutiques et Biologiques
> Université Paris Descartes
> 4, avenue de l'Observatoire
> 75006 PARIS
>
> tel : [phone]
> fax : [phone]
>   ----- Original Message -----
>   From: Shuchi Gupta
>   To: [hidden email]
>   Sent: Monday, March 09, 2009 5:23 PM
>   Subject: secondary antibody from immunostainigs
>
>
>   hello,
>     i want to know if somebody knows any good secondary antibodies(
>   alexa, atto conjugated) against rabbit which i can use for the
>   immunostainings. i have tried  antibodies from cell signaling, abnova
>   and dako.but none of them give me the specific signal .( this i get to
>   know from my controls which contain only my secondary antibody) .  i
>   am using these antibodies in mouse platelets.i block them with BSA 2%
>   after fixing and permeabilization.
>   hope to get the suggestions soon.
>   thank you
>   shuchi
>

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