Material and methods
Adult male or female canaries were anesthetized with 0.04 mL of Nembutal™ 0.6mg/mL and
perfused with 4% paraformaldehyde (Sigma) in phosphate buffered saline 0.1M (PBS pH 7.4
Sigma). Brains were removed from the skull, postfixed overnight in the same fixative
solution at 4¯C, rinsed in PBS, transferred to 30% sucrose in PBS and stored at 4¯C until they
sunk. They were frozen on dry ice and stored at -80¯C until used.
Brains were cut at 30 ´m thickness in the coronal plan and sections were collected in Trisbuffer
saline (TBS 0.05 M Tris, 0.9% NaCl, pH 7.6). Sections were transferred to a
cryoprotective solution (0.01M PBS with 10 g/L polyvinylpyrrolidone, 300 g/L sucrose, and
300 ml/L ethylene glycol) and stored at -20¯C until used.
Immunohistochemistry on free-floating sections
Sections were rinsed 3 X 5 minutes in TBS, 15 minutes in H2O2 3% in TBS, 3 X 5 minutes in
TBS and 30 minutes in blocking solution made of 1% bovine serum albumin, 0.1% triton X in
TBS. Sections were incubated in different dilutions of the primary antibody (1/500 1/2000
1/5000 in TBS- 0.1% triton-X 1% BSA) raised in rabbit against doublecortin (DCX ab18723) for
one hour at room temperature followed by overnight incubations for one or two nights at
4¯C with shaking.
Sections were then washed 3 X 5 minutes in TBS and incubated for 2 hours in the
secondary antibody solution at room temperature with shaking. This solution contained a
biotinylated goat anti-rabbit antibody (Jackson Immunoresearch 1/500) in TBS-0.1% triton-X
Sections were rinsed 3 X 5 minutes in TBS and incubated in the biotin-avidin complex
ABC (1/400 Vector Elite Kit, Vector Laboratories). The antigen-antibody complexes were
visualized thanks to a revelation kit (SG substrate kit for peroxydase, Vector laboratories).
Tissues were then mounted, dried and coverslipped with Eukitt mounting medium (Sigma).
The best dilution of the primary antibody was 1/2000 with a two nights incubation (Figure 1).
In sections stained in these conditions, the multipolar and fusiform DCX-positive neurons
were darkly stained and easy to distinguish from the background that was very minimal.
Counting of positive cells is thus very easy and positive cells are specifically present within
the anatomical landmarks where adult neurogenesis is known to take place.
On the contrary 1/500 generates a lot of background, which may hinder quantification. At
the 1/5000 dilution, the number of positive cells was lower in comparison with 1/2000
suggesting that this dilution is too high.
A good visualization of DCX neurons was obtained. Dense populations were namely
observed in the song control nucleus HVC and in the hippocampus.
Submitted Jul 13 2017
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