[Histonet] sheep/goat double labeling problem

[David A. Wright]

Hi Jonathan & Histonet
Apologies if you already got help here - I only read the
digest and didn't see a reply. Your problem is not with
"BrdU/Doublecortin double labeling " i.e. the epitopes but
with the hosts the Abs were raised in - sheep and goat.  These
are evolutionarily very close together and so their IgGs have
not diverged much in sequence. Consequently there is huge
interspecies cross reaction between anti-goat or anti-sheep
secondaries (which are polyclonal).  So the secondary (say,
anti-goat) against your second primary (made in goat) will
also bind in large part to the first primary (made in sheep) -
is that what you saw?.

You could look for secondaries that have been adsorbed against
the offending species, or you could use your existing reagents
and just include serum of the first primary-host species
(sheep, in the example) in with your second incubation
secondary mix (the anti-goat step in my example).  You will
lose a lot of activity that way and will probably need to up
your Ab concentration accordingly.  

But why don't you use more distinct hosts for your primaries?
 There are a plethora of anti-BrdU antibodies out there and
you can surely find something different from either goat or
your study species. If you are not working on a mouse tissues
with high endogenous IgGs, I'd recommend the BD MAb 347850. It
only needs a very simple denaturation procedure before the
primary:
Immerse the slides in 0.07 N NaOH for 2 minutes.
Immerse the slides in  PBS, pH 8.5, to neutralize the base.
- none of that cooking in HCl and/or formamide for hours.
The clone is B44 so you may find the same MAb from other
suppliers too.

-hope this helps

David Wright
Section of Neurosurgery
University of Chicago
==============================================
ORIGINAL MESSAGE
Message: 14
Date: Fri, 24 Aug 2007 10:25:32 -0400
From: "Jonathan Frank" <jfrank <@t> med.unc.edu>
Subject: [Histonet] BrdU/Doublecortin double labeling problem
To: histonet <@t> lists.utsouthwestern.edu
Message-ID: <200708241426.l7OEQ9br014226 <@t> smtp-mx.med.unc.edu>
Content-Type: text/plain; charset=us-ascii

Good morning.

I am attempting to double label Brain tissue with BrdU (Abcam
1893 raised in sheep) and Doublecortin (C-18 Santa Cruz raised
in goat) and I am having  some troubles that you may have
suggestions to.  The brains are perfused with 4%
paraformaldehyde and post fixed overnight at 4oC.  The brains
are then cryoprotected in 30% sucrose and cut on a cryostat at
20um.  I am using Alexa fluor 594 donkey-anti sheep and 488
donkey-anti goat for my fluorescent secondary antibodies.  I
have had good success using each primary/secondary
individually and the positive and negative controls seem
to act as expected.  The labeling also occurs in the correct
part of the cell individually.  The problem arises when I do
the double labeling step where any BrdU positive cell is also
doublecortin positive (not expected) and the morphology and
points of intensity are exactly the same.  I have tried this
on tissues where Doublecortin should not be expressed and come
up with the same problem.  Here is the protocol I have been using:

Blocking buffer = 8% donkey serum, 0.3% Bovine serum albumin,
0.3% Triton
X-100, in 1x PBS

Washing buffer = 0.3% Bovine serum albumin, 0.3% Triton X-100
in 1x PBS

1. 10mm Citrate buffer (pH 6) for 30' at 95oC
2. Allow to cool for 10'
3. 2 x 5' wash in 1x PBS
4. Block 1 hour
5. Doublecortin 1o AB (1:400 in washing buffer) overnight at 4oC
6. Wash 2 x 5' in washing buffer
7. 2N HCl 20' at RT
8. 2 x 5' wash in Tris Buffer (ph 7.6)
9. BrdU 1o AB (1:1500 in washing buffer) overnight at RT
10. 4 x 5' wash in washing buffer
11. Doublecortin 2o AB 1hr
12. Wash 2 x 5' in washing buffer
13. BrdU 2o AB 1hr
14. Wash 2 x 5' in Washing buffer
15. Wash 2 x 5' 1x PBS
16. Alcohols to Xylenes and coverslip

Has anyone encountered these problems or have suggestions as
to how I may
solve this problem?

Thanks in advance
Jon Frank

Jon Frank
Manager, Small Animal Resuscitation Research Lab
Department of Emergency Medicine
UNC-Chapel Hill
jfrank <@t> med.unc.edu
919-966-6442/919-966-7998