12 In Situ Hybridization for RNA: Nonradioactive Probe: Oligo-DNA Probe
177
Note: The appropriate dose of irradiation is 300-600 J/m 2 • 5000 JI
m 2 : irradiation 1000 secs with UV lamp (5 J Im 2 /sec). 10000 J/m 2 :
irradiation 2000 secs with UV lamp (5 J Im 2 /sec)
8. React the filter with a blocking solution for 1 hr at RT in a
vinyl bag. (250 IlI/l-2 filters)
9. React the filter with the blocking solution including HRP-Iabeled sheep anti-digoxigenin antibody (1: 400-BOO) for 3 hrs
in a vinyl bag. (250 1l1!1-2 filters)
10. Wash 4 times with 0.075% Brij 35/PBS for 15 mins each atRT.
11. After a brief washing with PBS, visualize the HRP sites in a
chromogen solution containing DAB, H20 2, nickel and cobalt at RT.
12. Wash the filter at least 3 times with DDW for 5 mins each and
dry up.
To confirm the specificity and the adequate sensitivity of probe Dot blot
oligo-DNAs, dot blot hybridization should be performed before hybridization
being processed to in situ hybridization study.
1. Prepare strips of nitrocellulose filters according to the steps
1-3 of the instruction for immunohistochemical detection.
2. Apply 2 III of DNA solution containing unlabeled oligoDNAs onto the filter at various quantities per dot (1 pg,
10 pg, 100 pg, 1 ng, 10 ng).
3. Dry up completely (overnight air-dry is recommended).
4. Bake the filter at BOoe for 2 hrs.
5. Irradiate the filter for 1 min.
6. Prehybridize the filter with a prehybridization mixture for
2 hrs at 37-42°e in a vinyl bag. (250 1l1!1-2 filters)
7. Hybridize the filter with a hybridization mixture for 15-17
hrs at 37-42°e in the vinyl bag. (250 1l1!1-2 filters)
8. Wash the filter successively as follows; 2 x sse (15 mins,
twice, RT), 1 x sse (15 mins, twice, RT), 0.5 x sse (15
mins, once, RT) and PBS (15 mins, once, RT).
177
Note: The appropriate dose of irradiation is 300-600 J/m 2 • 5000 JI
m 2 : irradiation 1000 secs with UV lamp (5 J Im 2 /sec). 10000 J/m 2 :
irradiation 2000 secs with UV lamp (5 J Im 2 /sec)
8. React the filter with a blocking solution for 1 hr at RT in a
vinyl bag. (250 IlI/l-2 filters)
9. React the filter with the blocking solution including HRP-Iabeled sheep anti-digoxigenin antibody (1: 400-BOO) for 3 hrs
in a vinyl bag. (250 1l1!1-2 filters)
10. Wash 4 times with 0.075% Brij 35/PBS for 15 mins each atRT.
11. After a brief washing with PBS, visualize the HRP sites in a
chromogen solution containing DAB, H20 2, nickel and cobalt at RT.
12. Wash the filter at least 3 times with DDW for 5 mins each and
dry up.
To confirm the specificity and the adequate sensitivity of probe Dot blot
oligo-DNAs, dot blot hybridization should be performed before hybridization
being processed to in situ hybridization study.
1. Prepare strips of nitrocellulose filters according to the steps
1-3 of the instruction for immunohistochemical detection.
2. Apply 2 III of DNA solution containing unlabeled oligoDNAs onto the filter at various quantities per dot (1 pg,
10 pg, 100 pg, 1 ng, 10 ng).
3. Dry up completely (overnight air-dry is recommended).
4. Bake the filter at BOoe for 2 hrs.
5. Irradiate the filter for 1 min.
6. Prehybridize the filter with a prehybridization mixture for
2 hrs at 37-42°e in a vinyl bag. (250 1l1!1-2 filters)
7. Hybridize the filter with a hybridization mixture for 15-17
hrs at 37-42°e in the vinyl bag. (250 1l1!1-2 filters)
8. Wash the filter successively as follows; 2 x sse (15 mins,
twice, RT), 1 x sse (15 mins, twice, RT), 0.5 x sse (15
mins, once, RT) and PBS (15 mins, once, RT).