SPAM-LOW: [Histonet] RE: Staining with two primary antibodies from same host

Patsy Ruegg pruegg <@t>
Tue Sep 8 07:27:03 CDT 2009

I would add an FC block before the first primary, I use one from Innovex for
20-30 min before the serum blocks, it is expensive but worth it for a mouse
on mouse detection system.


Patsy Ruegg, HT(ASCP)QIHC
12635 Montview Blvd. Ste.215
Aurora, CO 80045
fax 720-859-4110

-----Original Message-----
From: histonet-bounces <@t>
[mailto:histonet-bounces <@t>] On Behalf Of C.M. van der
Sent: Monday, September 07, 2009 1:36 AM
To: anonwums1 <@t>
Cc: histonet <@t>
Subject: SPAM-LOW: [Histonet] RE: Staining with two primary antibodies from
same host

Adam,Good point to prepare a protocol first, and then start staining! From
theoretical point of view your protocol should work. However, I have tried
this 'Jackson approach' using a Fab blocking step in a mouse-on-mouse
situation (before the MOM kits were available) without success. There are at
least two good solutions for double immunofluorescence using two primaries
from the same species:Brouns et al. JHC 50:575-582, 2002 and Uchihara et al.
JHC 51:1201-1206, 2003 applied a tyramide/fluorochrome detection for the
first primary and a simple two-step for the second. Because the tyramide
amplification is such a sensitive method, the first primary can be diluted
up to a level that the second simple two-step detection cannot pick up
signal from the first primary. Titration of the first primary antibody is
most important in this procedure. You can in vitro label your goat primary
with the Zenon (Invitrogen) kit based on anti-goat Fab fragments directly
labeled with an Alexa fluorochrome. Next, you can built up a multistep
indirect/direct double staining method:goat primary 1donkey
anti-goat/fluorochrome 1normal goat serum (1:10) for blockinggoat primary
2-Zenon in vitro labeled with anti-goat Fab/fluorochrome 2lots of success
with staining!ChrisChris van der Loos, PhD
Dept. of Pathology
Academic Medical Center M2-230
Meibergdreef 9
NL-1105 AZ Amsterdam
The Netherlands  Date: Fri, 4 Sep 2009 22:37:34 -0500
From: "Adam ." <anonwums1 <@t>>
Subject: [Histonet] Staining with two primary antibodies from same
To: histonet <@t>

Hi all,

I'm looking into staining with two primary antibodies from the same host, in
this case goat. I've read a bit about this on Jackson Immunoresearch's
website, but I wanted to run by my idea to get an idea if this is at all

I want to stain mouse tissue with antigen X and antigen Y. I have a two
polyclonal primaries: goat anti-mouse X and a goat anti-mouse Y. This is
what I was thinking

1) Block in donkey serum for 1 hr at room temp.
2) Incubate with goat anti-mouse X overnight at 4C. Wash.
3) Incubate with Dylight 488 donkey anti-goat for 30 mins at room temp.
4) Reblock in donkey serum for 1 hr at room temp.
5) Incubate with 10 - 20 ug / mL unconjugated donkey anti-goat Fab for 1 hr
at room temp -- cheapest I could find was at
6) Incubate with goat anti-mouse Y overnight at 4C. Wash.
7) Incubate with biotin donkey anti-goat for 30 mins at room temp. Wash.
8) Incubate with avidin AMCA for 30 mins at room temp

Would this work? Is there an easier or better way? What are the pitalls or
tips you could offer?

Hope you all aren't reading this during your long weekend,
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