[Histonet] Re: Rbt on Rbt

[David A. Wright]

Hi Karen (& Histonet)

Sorry for the long 'tutorial' here.

In principle you can make your own rabbit on rabbit pre-treatment before adding your primary.  The problem is specifically with any of the specimens's own IgG (rabbit, obviously) bound to your tissue. I haven't actually done it in rabbit but have dealt with the same problem in other rodent brains where any kind of damage (including the growth of tumors) makes the blood-brain barrier leaky so that the circulating IgG can seep out and bind to the brain tissue.

You have to 'block' all this leaked IgG before you apply the anti-rabbit fluorescent secondary. Since you don't want to block your primary as well, it has to be done before the primary too. You block with a cold (unlabelled) anti-rabbit IgG antibody - basically a secondary without its label.  How often are you going to do this?  If you're not going to do this often enough to purchase the cold anti-rabbit, you could use any non-fluorescent anti-rabbit secondary. (In my freezer I have biotinylated goat anti-rabbit which would do for this.)

Note that you have to cover up every last stray rabbit IgG, so you want to pre-incubate for much longer than your normal blocking step (with which it can be combined) - I would do it for as long as your normal primary incubation and/or at 2-5x your normal concentration for this non-fluorescent secondary. Don't forget to wash afterward, before the primary, or else you will neutralize your primary with the residue.

Sorry this isn't a specific protocol, but I haven't seen any other reply (I only read the daily digest)

-David
==
David A. Wright, Ph.D.
University of Chicago
Section of Neurosurgery, MC3026

>Message: 1
>Date: Thu, 1 Dec 2011 10:01:31 -0800
>From: "Karen Cai" <kcai <@t> prosci-inc.com>
>Subject: [Histonet] Rabbit antibody on rabbit tissue


Hi All,

I am going to apply one rabbit polyclonal antibody on the rabbit tissue using IF. Is there anybody do it before? I heard of mouse on mouse IHC-DAB method, but not sure if it's possible in IF.
 
Your kind response is very appreciated,
Thanks,
Karen