24
SHIGEYOSHI OBA AND TAKEHIKO KOJI
Procedure
Paraffin sections with biotin-dUTP
1. Samples are fixed in 4% paraformaldehyde in phosphatebuffered saline (pBS), pH 1.4 at RT about 4-lOhrs.
2. Samples are rinsed with PBS 3times for 5mins each, dehydrated and embedded in paraffin block by routine method.
3. Cut sections to a thickness of 5-6)Jm and place them on
silane-coated glass slides according routine procedure.
4. Deparaffinize the sections with toluene 3times (5 mins in
each cup), and rehydrate through graded ethanol.
5. Wash with distilled water for 5 mins, 2 times, then immerse
in PBS for 5 mins.
6. Proteinase K treatment; Incubate the sections in the proteinase K solution(O.5 -lO)Jg/ml in PBS) at 37°C for 15mins.
Note: You must change the concentration of proteinase K according to sample and fixation condition. You had better try several concentration of proteinaseK for first time(for example 0.1,
0.5, 1, 5, lO)Jg/ml) and decide on the most appropriate concentration for your sample.
(Put the sections into the cup in 37°C water bath, which contains
100)J1-2ml PK(500)Jg/ml in PBS) and 100mi PBS.)
Note: You had better stock higher concentration of proteinase
K(100 or 500)Jg/ml in PBS) below -20°e.
7. Wash with PBS 3 times,S mins each
8. Rinse once with distilled water.
9. Preincubate with 1 x TdT buffer, which contains 0.2M potassium cacodylate, 25 mM Tris-HCI(pH 6.6) and 0.025%BSA
for 30mins at room temperature(RT). (Tap off excess water,
carefully wipe around specimen and apply TdT buffer to
cover specimen in moist chamber)
10. Incubate in Ix TdT buffer at 37°C for 2hrs in moist chamber.
SHIGEYOSHI OBA AND TAKEHIKO KOJI
Procedure
Paraffin sections with biotin-dUTP
1. Samples are fixed in 4% paraformaldehyde in phosphatebuffered saline (pBS), pH 1.4 at RT about 4-lOhrs.
2. Samples are rinsed with PBS 3times for 5mins each, dehydrated and embedded in paraffin block by routine method.
3. Cut sections to a thickness of 5-6)Jm and place them on
silane-coated glass slides according routine procedure.
4. Deparaffinize the sections with toluene 3times (5 mins in
each cup), and rehydrate through graded ethanol.
5. Wash with distilled water for 5 mins, 2 times, then immerse
in PBS for 5 mins.
6. Proteinase K treatment; Incubate the sections in the proteinase K solution(O.5 -lO)Jg/ml in PBS) at 37°C for 15mins.
Note: You must change the concentration of proteinase K according to sample and fixation condition. You had better try several concentration of proteinaseK for first time(for example 0.1,
0.5, 1, 5, lO)Jg/ml) and decide on the most appropriate concentration for your sample.
(Put the sections into the cup in 37°C water bath, which contains
100)J1-2ml PK(500)Jg/ml in PBS) and 100mi PBS.)
Note: You had better stock higher concentration of proteinase
K(100 or 500)Jg/ml in PBS) below -20°e.
7. Wash with PBS 3 times,S mins each
8. Rinse once with distilled water.
9. Preincubate with 1 x TdT buffer, which contains 0.2M potassium cacodylate, 25 mM Tris-HCI(pH 6.6) and 0.025%BSA
for 30mins at room temperature(RT). (Tap off excess water,
carefully wipe around specimen and apply TdT buffer to
cover specimen in moist chamber)
10. Incubate in Ix TdT buffer at 37°C for 2hrs in moist chamber.