12 In Situ Hybridization for RNA: Nonradioactive Probe: Oligo-DNA Probe
179
6. Fix in 4% PF A/PBS for 5 mins and wash twice with PBS.
7. Immerse twice in 2 mg/ml glycine/PBS for 15 mins each.
8. After brief washings with PBS and DDW, successively, immerse in 40% deionized formamide/4 x sse until used for
hybridization.
9. Put 20-30 IJI of the hybridization mixture on each slide and
keep it in a moist chamber humidified with 40% formamide/
4 x sse at 37°e for 15-17 hrs. Under these conditions, no
covers to avoid evaporation of the medium are needed.
Note: Furthermore, the addition of 200 U/ml heparin and/or
0.01 % SDS to the hybridization mixture often decreased the nonspecific reaction of the probes.
10. Wash 5 times with 0.075% Brij 35/2 x sse for 1 hr each time at
37 °e and twice with 2 x sse for 15 mins each.
Note: If necessary, 50% formamide is included, and for a more
stringent washing lower concentrations of salt such as 0.5 x sse
and 0.2 x sse are used.
11. Immerse in 70% ethanol (5 mins), 90% ethanol (5 mins),
100% ethanol (5 mins, twice), and acetone (5 mins, twice),
successively, as a delipidation.
Note: This step after hybridization is effective in removing the
probe bound to sections nonspecifically (Koji et al. 1994), especially in some cases including decalcified tissues (Iwasaki et
a1.1998).
12. After a brief washing with PBS, put 30-40 IJI of a blocking
solution and leave to stand for 1 hr in a moist chamber humidified with PBS.
13. React with 30-40 IJI of the blocking solution including HRPlabeled sheep anti-digoxigenin antibody (1: 100-200) for 1517 hrs in the moist chamber.
14. Wash 4 times with 0.075% Brij 35/PBS for 15 mins each.
15. After a brief washing with PBS, visualize the HRP sites in a
chromogen solution containingDAB,HzOz, nickel and cobalt.
16. Dehydrate with serial up-graded ethanol solutions, cleared
with xylene and mounted without any counterstaining.
179
6. Fix in 4% PF A/PBS for 5 mins and wash twice with PBS.
7. Immerse twice in 2 mg/ml glycine/PBS for 15 mins each.
8. After brief washings with PBS and DDW, successively, immerse in 40% deionized formamide/4 x sse until used for
hybridization.
9. Put 20-30 IJI of the hybridization mixture on each slide and
keep it in a moist chamber humidified with 40% formamide/
4 x sse at 37°e for 15-17 hrs. Under these conditions, no
covers to avoid evaporation of the medium are needed.
Note: Furthermore, the addition of 200 U/ml heparin and/or
0.01 % SDS to the hybridization mixture often decreased the nonspecific reaction of the probes.
10. Wash 5 times with 0.075% Brij 35/2 x sse for 1 hr each time at
37 °e and twice with 2 x sse for 15 mins each.
Note: If necessary, 50% formamide is included, and for a more
stringent washing lower concentrations of salt such as 0.5 x sse
and 0.2 x sse are used.
11. Immerse in 70% ethanol (5 mins), 90% ethanol (5 mins),
100% ethanol (5 mins, twice), and acetone (5 mins, twice),
successively, as a delipidation.
Note: This step after hybridization is effective in removing the
probe bound to sections nonspecifically (Koji et al. 1994), especially in some cases including decalcified tissues (Iwasaki et
a1.1998).
12. After a brief washing with PBS, put 30-40 IJI of a blocking
solution and leave to stand for 1 hr in a moist chamber humidified with PBS.
13. React with 30-40 IJI of the blocking solution including HRPlabeled sheep anti-digoxigenin antibody (1: 100-200) for 1517 hrs in the moist chamber.
14. Wash 4 times with 0.075% Brij 35/PBS for 15 mins each.
15. After a brief washing with PBS, visualize the HRP sites in a
chromogen solution containingDAB,HzOz, nickel and cobalt.
16. Dehydrate with serial up-graded ethanol solutions, cleared
with xylene and mounted without any counterstaining.