Immunohistochemistry

Immunohistochemistry

1. Anesthetize mice by intraperitoneal injection of 10% somnopentyl (1.0 mg/kg, Kyoritsu Seiyaku Corporation, Tokyo, Japan)

2. Transcardially perfuse mice first with 20 ml of ice-cold saline. This perfusion was immediately followed by another 20 ml of 10% ice-cold formalin (Wako Pure Chemical Industries, Ltd., Osaka, Japan)

3. Dissect out brains and post-fix in 10% ice-cold formalin at 4⁰C overnight

4. Brains were then subsequently immersed in 30% sucrose in PBS at 4°C for at least 2 days

5. Embed brains in O.C.T. Compound and freeze at -80⁰C. Brains can be stored at this stage until required

6. Glue brain in the stage of cryostat (Leica CM3050 S; Leica Microsystems, Wetzlar, Germany) and cut 40µm coronal sections of the desired brain regions 

7. Carefully collect sections with a paintbrush and move to wells of 24 well plate containing PBS (Brain slices can be stored at this stage 4°C until required)

8. Transfer sections with a brush to 24 well plate and wash 3 times by the buffer solution PBS-BX

9. Incubate the brain slices with primary antibodies at 4°C overnight in the 24 well plate

10. The brain slices were washed with blocking buffer for 3 times

11. Incubate the brain slices with secondary antibodies for 1 hr at room temperature (RT). 

12. The brain slices were washed with blocking buffer for 3 times

13. Brain sections were mounted on a glass slide and wait until dry at room temperature in a dark condition

14. Examined glass slide containing the brain sections using a fluorescence microscope (BZ-9000, Keyence, Osaka, Japan or IX71, Olympus, Tokyo, Japan)

Solutions:

1. Blocking buffer: PBS-BX (1% BSA and 0.25% Triton-X in PBS)

Tested primary antibodies:

How to cite: Readers should cite both the Bio-protocol article and the original research article where this protocol was used: 

1. Chowdhury, S. (2020). Immunohistochemical staining - Bio-protocol.bio-protocol.org/Rap11574 

2. Chowdhury S, Hung CJ, Izawa S, Inutsuka A, Kawamura M, Kawashima T, Bito H, Imayoshi I, Abe M, Sakimura K, Yamanaka A. Dissociating orexin-dependent and -independent functions of orexin neurons using novel Orexin-Flp knock-in mice. eLife. 2019;8. doi:10.7554/eLife.44927

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