Immunohistochemistry

Fluorescence:

Day 1

1. 3X10 min washes in 0.1 M PB.
2. 1% H2O2 in 0.1 M PB – 15 min (i.e. 30 uL of 30 % H2O2 /mL PB = 3.6 mL H2O2 in 120 mL 0.1 M PB)
3. 3X10 min washes in 0.1 M PB
4. Transfer tissue to 4% NHS (normal horse serum) in 0.1 M PB + 0.3% Triton X-100 for 30 minutes (4.8ml NHS in 120ml of 0.1 M PB + triton).
5. Dilute primary antibody in 0.1 M PB + 0.3% Triton X-100 + 4% NHS
6. Incubate tissue overnight at 4 C.

 Day 2

1. 3x10 min washes in  0.1 M PB  
2. Dilute secondary antibody in 0.1 M PB (1:400)
3. Incubate for 90 minutes room temperature. Cover with alufoil or 
4. Wash in 0.1 M PB 3x10 minutes. 
5. Begin mounting on super frost slides (without gel coating).
6. Coverslip straight after mounting, but ensure that the slide are mostly dry (if not your sections will move around when placing the coverslip.
7. Use fluorescent mounting medium (DAKO)
8. Once mounted, seal the coverslip to the slide with clear nailpolish.