136
HIROTAKA MATSUMI AND TETSU YANO
In situ hybridization (indirect method)
1. Cut sections to a thickness of 5-6 11m and place them on silane-coated glass slides according to the routine procedure.
2. Prior to use, warm at 60°C for at least 30 mins, deparaffinize
through toluene and then rehydrate with serial ethanol solution and wash 3 times with PBS for 5 mins each.
3. Immerse in 0.3 % H20 2 in methanol for 15 mins to block
endogenous peroxidase activity and wash 2 times with
PBS for 5 mins each.
4. After a brief washing with D DW, immerse in 0.2 N H Cl for 20
mins and wash successively with DDW.
5. Treat with 0.2 % Triton X-100 in PBS, shaking gently, for 10
mins and wash with PBS for 5 mins.
6. Treat with proteinase K (1-100 Ilg/ml in PBS) at 37°C for 15
mins and wash 3 times with PBS for 5 mins each.
7. Immerse in 4 % PF A/PBS for 5 mins to postfix the target
nucleic acid.
8. Immerse twice in 2 mg/ml glycine/PBS for 15 mins each to
remove the PF A.
9. After briefly washing with PBS and DDW, successively, immerse in 40 % deionized formamide/4x SSC until used for
hybridization (more than 30 mins).
10. Wipe each slide around the tissue and put 30-40 III of the
hybridization mixture on each slide and keep it in a moist
chamber humidified with 40 % deionized formamide/4x SSC
at 37 DC for 12 hrs. Under these conditions, no covers to
avoid evaporation of the medium are needed.
11. Wash 5 times with 50 % deionized formamide/0.5x SSC at
37°C for Ihr each time and twice at 45 DC for 15 mins
each and twice with 2x SSC for 15 mins each.
12. After washing with PBS for 5 mins, drain each slide with a
gause, put 30-40 /!l of the blocking solution on each slide and
leave to stand for 1 h in a moist chamber humidified with
PBS.
HIROTAKA MATSUMI AND TETSU YANO
In situ hybridization (indirect method)
1. Cut sections to a thickness of 5-6 11m and place them on silane-coated glass slides according to the routine procedure.
2. Prior to use, warm at 60°C for at least 30 mins, deparaffinize
through toluene and then rehydrate with serial ethanol solution and wash 3 times with PBS for 5 mins each.
3. Immerse in 0.3 % H20 2 in methanol for 15 mins to block
endogenous peroxidase activity and wash 2 times with
PBS for 5 mins each.
4. After a brief washing with D DW, immerse in 0.2 N H Cl for 20
mins and wash successively with DDW.
5. Treat with 0.2 % Triton X-100 in PBS, shaking gently, for 10
mins and wash with PBS for 5 mins.
6. Treat with proteinase K (1-100 Ilg/ml in PBS) at 37°C for 15
mins and wash 3 times with PBS for 5 mins each.
7. Immerse in 4 % PF A/PBS for 5 mins to postfix the target
nucleic acid.
8. Immerse twice in 2 mg/ml glycine/PBS for 15 mins each to
remove the PF A.
9. After briefly washing with PBS and DDW, successively, immerse in 40 % deionized formamide/4x SSC until used for
hybridization (more than 30 mins).
10. Wipe each slide around the tissue and put 30-40 III of the
hybridization mixture on each slide and keep it in a moist
chamber humidified with 40 % deionized formamide/4x SSC
at 37 DC for 12 hrs. Under these conditions, no covers to
avoid evaporation of the medium are needed.
11. Wash 5 times with 50 % deionized formamide/0.5x SSC at
37°C for Ihr each time and twice at 45 DC for 15 mins
each and twice with 2x SSC for 15 mins each.
12. After washing with PBS for 5 mins, drain each slide with a
gause, put 30-40 /!l of the blocking solution on each slide and
leave to stand for 1 h in a moist chamber humidified with
PBS.