Advanced Search
Immunohistochemistry
Anesthetize mice by intraperitoneal injection of 10% somnopentyl (1.0 mg/kg, Kyoritsu Seiyaku Corporation, Tokyo, Japan)
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)
Dissect out brains and post-fix in 10% ice-cold formalin at 4⁰C overnight
Brains were then subsequently immersed in 30% sucrose in PBS at 4°C for at least 2 days
Embed brains in O.C.T. Compound and freeze at -80⁰C. Brains can be stored at this stage until required
Glue brain in the stage of cryostat (Leica CM3050 S; Leica Microsystems, Wetzlar, Germany) and cut 40µm coronal sections of the desired brain regions
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)
Transfer sections with a brush to 24 well plate and wash 3 times by the buffer solution PBS-BX
Incubate the brain slices with primary antibodies at 4°C overnight in the 24 well plate
The brain slices were washed with blocking buffer for 3 times
Incubate the brain slices with secondary antibodies for 1 hr at room temperature (RT).
The brain slices were washed with blocking buffer for 3 times
Brain sections were mounted on a glass slide and wait until dry at room temperature in a dark condition
Examined glass slide containing the brain sections using a fluorescence microscope (BZ-9000, Keyence, Osaka, Japan or IX71, Olympus, Tokyo, Japan)
Solutions:
Blocking buffer: PBS-BX (1% BSA and 0.25% Triton-X in PBS)
Tested primary antibodies:
Antibody | Maker | Cat# | Dilution |
goat polyclonal anti-orexin | Santa Cruz Biotechnology | sc-8070 | (1/1000) |
rabbit polyclonal anti-MCH | Sigma-Aldrich | M8440 | (1/2000) |
rabbit polyclonal anti-prodynorphin | Merck Millipore | AB5519 | (1/100) |
mouse monoclonal anti-c-Fos | Santa Cruz Biotechnology | E-8 | (1/500) |
mouse monoclonal anti-DsRED | Santa Cruz Biotechnology | sc-390909 | (1/1000) |
mouse monoclonal anti-GFP | Fujifilm Wako Pure Chemical Corporation | mFX75 | (1/1000) |
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
Copyright: Content may be subjected to copyright.
Do you have any questions about this protocol?
Post your question to gather feedback from the community. We will also invite the authors of this article to respond.
Tips for asking effective questions
+ Description
Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images.
Share
Bluesky
X
Copy link