[Histonet] double trouble

Andrea Hooper anh2006 <@t> med.cornell.edu
Mon Nov 3 10:21:45 CST 2008

You cannot do what Paula recommends below without extra blocking 
steps, if the primaries are raised in the same species as the second 
secondary is raised. This is a rough outline of what I recommend:

1st primary antibody (X-anti-Y)
1st biotinylated secondary antibody (Fab anti-X)
H202 block (to block HRP)
Avidin-biotin blocking kit (to block biotin from 1st secondary)
Block with excess unlabeled Fab anti-X (to block any sites on 1st 
primary which might be bound by 2nd secondary)
2nd primary antibody (X anti-Z)
2nd biotinylated secondary antibody (Whole IgG anti-X - could also 
use Fab, it's just unnecessary/more expensive)
Another HRP-based chromagen of your choosing (True Blue, Vector VIP, AEC+ etc)

This can be made simpler by doing HRP and AP but I am not a fan of AP 
so I cannot personally recommend that.

Good luck ... and the new protocol Gudrun mentioned that Chris 
published sounds very interesting!

At 6:23 AM -0800 11/3/08, Paula Pierce wrote:
>Incubate with one antibody and use DAB as the chromagen, then go 
>back and incubate with the second antibody and use a colored 
>chromagen such as AEC. Paula


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