Immunohistochemistry (PFA perfusion fixed frozen sections) abreview for Anti-Doublecortin antibody

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Abreviews
Application
Immunohistochemistry (PFA perfusion fixed frozen sections)
Sample
Bird Tissue sections (Serinus canaria forma domestica brain)
Antigen retrieval step
None
Permeabilization
Yes - triton-x 0,1% in Tris buffered saline
Specification
Serinus canaria forma domestica brain
Blocking step
BSA as blocking agent for 30 minute(s) · Concentration: 1% · Temperature: 24°C
Fixative
Paraformaldehyde

Other product details

Incubation time
48 hour(s) and 0 minute(s) · Temperature: 4°C
Dilution
1/2000

Secondary antibody

Dilution
1/500
Name
Non-Abcam antibody was used: oat anti-rabbit biotin-sp-conjug
Host species: Goat
Clonality: Polyclonal
Conjugation: Biotin

Additional data

Additional Notes
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 1% BSA. 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). Results 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. Conclusion A good visualization of DCX neurons was obtained. Dense populations were namely observed in the song control nucleus HVC and in the hippocampus.

Laura Vandries

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Submitted Jul 13 2017

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