3 In Situ Nick End-Labeling: Light Microscopical
21
Air dry cryostat sections(5-8Ilm)
PBS 5mins
Fix with 4% paraformaldehyde 20mins
-lPBS 5minsX3
-lProteinase K l5mins at 37°C
PBS 5minsX3
-lPreincubate with TdT buffer RT 30mins
TdT reaction 37°C 2hrs
Tris HCl rinse 5minsX3
0.3% Hz02 RT 5mins
Rinse BrijlPBS 5minsX3
-lNormal sheep IgG RT Ihr
-lHRP-conjugated sheep anti-digoxigenin RT 2hrs
-lRinse BrijlPBS lOminsX4
-lDAB-H202 with CoC12 and NiSOiNH4hS04 RT 5mins
-lRinse distilled water> 5mins
Dehydration and mounting
Fig. 2. Frozen sections with digoxigenin-ll-dUTP