134
HIROTAKA MATSUMI AND TETSU YANO
In situ hybridization (direct method)
1. eut sections 5-6 f.lm thick and place them on silane-coated
glass slides according to routine procedure.
2. Prior to use, warm at 60 °e 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 DDW, immerse in 0.2 N Hel 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 f.lg/ml in PBS) at 37 e for 15
mins and wash 3 times with PBS for 5 mins each.
7. Immerse in 4 % PFA/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/4 x sse until used for
hybridization (more than 30 mins).
10. Wipe each slide around the tissue and put 30-40 f.ll of the
hybridization mixture on each slide and keep it in a moist
chamber humidifIed with 40 % deionized formamide/4 x
sse at 37 °e 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 sse at 37
°e for Ihr each and twice at 45 °e for 15 mins each and twice
with 2x sse for 15 mins each.
12. After washing with PBS for 5 mins, drain each slide with a
gause, put 30-40 f.ll 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 (direct method)
1. eut sections 5-6 f.lm thick and place them on silane-coated
glass slides according to routine procedure.
2. Prior to use, warm at 60 °e 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 DDW, immerse in 0.2 N Hel 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 f.lg/ml in PBS) at 37 e for 15
mins and wash 3 times with PBS for 5 mins each.
7. Immerse in 4 % PFA/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/4 x sse until used for
hybridization (more than 30 mins).
10. Wipe each slide around the tissue and put 30-40 f.ll of the
hybridization mixture on each slide and keep it in a moist
chamber humidifIed with 40 % deionized formamide/4 x
sse at 37 °e 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 sse at 37
°e for Ihr each and twice at 45 °e for 15 mins each and twice
with 2x sse for 15 mins each.
12. After washing with PBS for 5 mins, drain each slide with a
gause, put 30-40 f.ll of the blocking solution on each slide and
leave to stand for 1 h in a moist chamber humidifIed with
PBS.