Immunofluorescence staining

Immunofluorescent staining on free-floating mouse brain sections

1. Transfer free-floating sections from cryoprotection solution into phosphate buffered saline (PBS) provided in a 12-well plate.
2. Wash sections twice with PBS (10 min each wash).
3. (Only BrdU staining): Wash sections twice with 0.9 % sodium chloride (5 min each wash).
4. (Only BrdU staining): Incubate sections in 2 M HCl for 30 min at 37 °C.
5. (Only BrdU staining): Wash sections 4 times with PBS (10 min each wash).
6. Incubate sections in blocking solution (PBS supplemented with 10 % normal donkey serum, Jackson ImmunoResearch, and 0.2 % TritonX-100, Merck) for 1.5 h at room temperature.
7. Incubate sections with primary antibodies diluted in antibody solution (PBS supplemented with 3 % donkey serum and 0.2 % TritonX-100) overnight at 4 °C.
8. Remove antibody solution and wash sections three times with PBS (10 min each wash).
9. Incubate sections with secondary antibody (e. g. Cy3 donkey anti-rat; Jackson ImmunoResearch) diluted in antibody solution (1:1000) for 2 h at room temperature.
10. Counterstain nuclei with 4′,6-diamidino-2-phenylindole (DAPI; 3.3 µg/ml diluted in PBS) for 10 min at room temperature
11. Wash sections twice with PBS (10 min each wash).
12. Transfer sections into 0.1 M phosphate buffer and mount on glass slides. 
13. Cover sections with mounting medium (e. g. Aqua-Poly/Mount; Polysciences) and coverslips. 
14. Store stained sections at 4 °C.

Recommended primary antibodies: 

Anti-Ki67 (polyclonal rat; eBioscience: 14-5698-82; dilution 1:500)

Anti-BrdU (monoclonal rat; AbD Serotec: OBT0030; dilution 1:500)

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