- 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].
- 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.
- Wash (3 x 15 min) in 0.1M PB/0.3% triton*.
- Preincubate in 0.1M PB/0.3% triton/1% blocking serum** for at least 1 h.
- Incubate at 4oC in primary antibody
- Wash (3 x 15 min) in 0.1 M PB/0.3% triton.
- 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].
- Wash (3 x 15 min) in 0.1M PB. [Note: No triton].
- Wash once in 0.1M acetate buffer - briefly (just to remove PB). [Acetate buffer must be made up fresh].
- Place sections in solution for DAB reaction (see below). Monitor DAB reaction on microscope (2-10 min).
- 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.
Glucose oxidase-nickel-DAB method (adapted from Shu et al).
- 0.2M acetate buffer: 3.28 g in 200 ml ddH2O. Adjust to pH6 with acetic acid.
- In 50 ml 0.2 M acetate buffer dissolve 2.5 g of nickel sulphate. (100 ml beaker).
- Add in this order: 200 mg glucose, 40 mg ammonium chloride, 50 ml DAB solution (0.5 mg/ml) - filter into 100 ml beaker, 1.5 mg glucose oxidase
Mount sections on gelatinised twin-frosted slides and dry in oven overnight or air dry on bench. Dehydrate with histoclear and ethanol (30%, 70%, 95%). Coverslip with Ralmount.Use fluoromount for Fluorescent sections.
For 1L of 0.2M phosphate buffer:
6.24g of NaH2PO4.2H2O or 5.52g of NaH2PO4.H2O
28.48 of Na2HPO4.2H2O or 22.72g of Na2HPO4 pH @ 7.4