[Histonet] BrdU/Doublecortin double labeling problem

[Jonathan Frank]

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