26
SHIGEYOSHI OBA AND TAKEHIKO KOJI
TdT reaction
solution -
digoxigenin
Frozen section with digoxigenin-dUTP
1. Cryostat sections (5-8 11m) are cut from snap-frozen tissue
blocks. and air dried for 2-4hours.
2. Wash dried sections with PBS and fix in the 4% paraformaldehyde in PBS for 20 mins at R T.
3. Wash the samples with PBS for 3 times,S min each
4. Proteinase K treatment; Incubation the sections in the Proteinase K solution(0.5-l.0Ilg/ml in PBS) at 37°C for 15min.
Note: You had better use lower concentration of proteinaseK for
frozen sections than paraffin sections.
5. Wash with PBS 3 times, 5 mins each
6. Rinse once with distilled water.
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12.
Preincubate with 1 x T dT buffer, for 30mins at room temperature(RT).
Incubate in TdT reaction solution -digoxigenin 37°C for 2hrs
in moist chamber.
Rinse the samples 3 times with 50mM PH 7.5 Tris-HCI for
5 mins each.
Immerse the samples in 0.3% H20 2 in methanol for 15 mins
at RT to inactivate the endogeneous peroxidase.
Wash three times with Brij/PBS for 5 mins each.
After a brief washing with PBS, incubate with normal sheep
IgG(O.5 mg/ml in 5% BSA/PBS) at RT for Ihr to reduce the
nonspecific binding of antibody to the samples.
13. Incubate with HRP-conjugated sheep anti-digoxigenin
(1/100 in 1 %BSA/PBS) at RT for 2hrs.
14. Wash with 0.075% Brij/PBS 4 times, 10 mins each, then immerse in PBS.
15. Visualize with DAB-HzOz and enhance with CoClz and
NiS04(NH4hS04 at RT for 5mins.
16. Wash with running water for short time and finally distilled
water for 5 mins.
17. Dehydration and mounting.