10 In Situ Hybridization for RNA: Nonradioactive Probe: ss eDNA Probe
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2. Incubate 10 mins at 37°C
3. Incubate 10 mins in boiling water.
4. Confirm that the sizes of DNA fragment are about 100-300
bases by agarose gel electrophoresis.
Here I present an example of one of the possible protocols. See
Chapter 11, for further explanation.
1. Place the frozen sections (4-10 urn in thickness) on silanecoated glass slides.
2. Dry quickly using hair-dryer with cool air.
3. Fixed with 4% PF A in PBS for 5 mins at RT, wash in PBS 5 mins
and wash in DDW 5 mins.
4. Dip in 0.2N HCl for 20 mins RT and wash in PBS 5 mins two
times.
5. Dip in 1 /-1g/ml proteinase K for 15 mins 37°C and wash in PBS
5 mins two times.
6. Dip in 40% formamide in 4X SSC RT 30 mins.
7. Mix the following as hybridization solution (l00 /-11):
1 M Tris-HCl
1 J.!l
5 M NaCI
12 J.!l
200 mM EDTA
0.5 )ll
100X Denhardt'
1 )ll
5 mg/ml ssDNA
2 J.!l
10 mg/ml tRNA
2 )ll
DDW
39 J.!l
Deionized formamide
40 J.!l
44 J.!g/ml ss eDNA probe
2.3 III
8. Heat up the hybridization solution in boiling water for 3 mins,
and chill in ice water.
9. Put the hybridization solution on the sections.
In situ hybridization