实验概要
The method provides a IHC protocol for free floating brain sections.
实验步骤
1. Coronal 30-40 µm sections cut on a freezing microtome. Sections collected into petri dishes containing 1-2ml 0.1M phosphate buffer (PB). Sections can be kept on a shaker at 4oC for several days before commencing the immunocytochemistry. [If left too long, the sections become harder to mount].
2. Deactivate endogenous peroxidases [For 40 ml: 20 ml 0.2M PB, 8 ml methanol, 80 µl triton-X100, 2 ml hydrogen peroxide and make up to 40 ml with ddH2O]. 15 min incubation.
3. Wash (3 x 15 min) in 0.1M PB/0.3% triton*.
4. Preincubate in 0.1M PB/0.3% triton/1% blocking serum** for at least 1 h.
5. Incubate at 4oC in primary antibody
6. Wash (3 x 15 min) in 0.1 M PB/0.3% triton.
7. Secondary antibody either 2h, room temperature (1:200) or overnight at 4oC @ 1:500 [made up in buffer 0.1M PB/0.3% triton/1% blocking serum].
8. Wash (3 x 15 min) in 0.1M PB. [Note: No triton].
9. Wash once in 0.1M acetate buffer - briefly (just to remove PB). [Acetate buffer must be made up fresh].
10. Place sections in solution for DAB reaction (see below). Monitor DAB reaction on microscope (2-10 min).
11. Stop reaction once background is high enough by placing sections into 0.1M acetate buffer. Finally, 3 washes in 0.1M PB. Sections can be kept in cold room on share for a couple of days, or until mounted and cover-slipped.
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