[Histonet] BrdU/DCX problem

[Jonathan Frank]

Dear list,

 

I am trying to double label cells with BrdU and Doublecortin (DCX) in the
adult rat brain and I am running into some issues.  I have been able to get
both antibodies to work well individually and together in the same tissue
but I am not getting the co-labeling of cells that the literature supports.
Our naïve untreated rats do not show any co-labeling of DCX and BrdU while
the literature shows that there should be quite a bit of this occurring.
Since BrdU is nuclear and DCX is cytoplasmic I do not believe that one is
blocking the other but maybe I am forgetting something.  I was wondering if
anyone has used BrdU/DCX and have had similar problems or if there is
something that I am missing.  Below are some details of the protocol.

 

Animal:  Adult female Sprague dawley rats (~275g)

Tissue: perfused and fixed in 4% paraformaldehyde (overnight) and frozen in
liquid nitrogen

Slide prep: 10um sections

Primary AB’s:    Abcam polyclonal rabbit anti-DCX (ab 18723)

                        Abcam polyclonal sheep anti-BrdU (ab 1893)

 

Secondary detection: Alexa Fluor donkey anti-sheep

                               Alexa Fluor goat anti- rabbit

 

1.	Incubate slides for 30’ in Citrate buffer pH 6.0 at 95oC in water
bath.  Place citrate buffer in plastic slide cover and then into water bath.
Set water bath to 95oC.
2.	Allow slides to cool in citrate buffer for 10’
3.	Wash 2 x 5’ in 1x PBS
4.	Place 2-3 drops of 2N HCl on each slide and incubate for 20’ at room
temperature.
5.	Wash 2 x 5’ in 1x PBS
6.	Wash with 0.1M Tris buffer 2 x 5 minutes
7.	Wash 2 x 5’ in 1x PBS
8.	Block with 10% normal donkey serum in washing buffer for 60’ 
9.	Incubate in primary BrdU antibody overnight at room temperature
(dilution: 1.5uL/mL, or 5ug/mL).

a.       Make necessary amount of BrdU antibody (1.5:1000 dilution in
washing buffer).

10.	Gently tap off BrdU primary antibody.
11.	Wash 5 x 5’ in washing buffer.  
12.	Incubate for 60 minutes in secondary antibody for BrdU (dilution
2ul/1mL, or 1:500)
13.	Wash 2 x 5’ in washing buffer
14.	Wash 2 x 5’ in 1x PBS

15.  Block for 1 hour in blocking buffer (8% goat serum, 0.3% Bovine serum
albumin, 0.3% Triton X-100, in 1x PBS) at room temperature.  For DCX

16.  Incubate with DCX primary antibody (1:1000 dilution in washing buffer)
overnight at room temperature

a.       Make necessary amount of DCX (1:1000 dilution in washing buffer)

17.  Wash 5 times for 5 minutes each with washing buffer. Tap excess buffer
off of slide.

18.  Add secondary antibody (AF goat anti-rabbit 488 1:500 in washing
buffer) and incubate for 60 minutes at room temperature.  

19.  Wash 2 x 5’ in washing buffer

20.	Wash once for 5 minutes in distilled water.
21.	Coverslip 
22.	Store in cool dark place (refrigerator)

 

 

 

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