11 In Situ Hybridization for RNA: Nonradioactive Probe: Oligo-DNA Probe
161
Immunohistochemical detection
1. Cut lx4 cm strips of nitrocellulose filters and immerse in
DDW for 30 mins at RT.
2. Replace DDW for 20x SSC and keep them for 30 mins at RT.
3. Place the filters on a sheet of Parafilm and dry up.
4. Apply 2 /ll of DNA solution containing DNA at various quantities per dot (10 ng,1 ng,100 pg, 10 pg, 1 pg).
5. Dry up completely (overnight air-dry is recommended).
6. Bake the filter at 80°C for 2 hrs exactly in an oven.
7. Place the filter inside a vinyl bag and incubate with a blocking solution consisting of 5% BSA (98-99% pure), 500 /lg/ml
normal goat IgG, 100 /lg/ml yeast tRNA, 100 /lg/ml salmon
sperm DNA, (0.05% NaN3) and PBS, for Ihr at RT.
8. Take the filter out of bag and replace the blocking solution
with the 1st antibody (mouse anti-T -T IgG; 1:80) dissolved in
PBS containing 5% BSA, 100 /lg/ml yeast tRNA, 100 /lg/ml
salmon sperm DNA and 0.05% NaN3, for 3 hrs at RT in a
vinyl bag.
9. Wash the filter with 0.075% Brij 35 in PBS at RT (4x 15 mins).
10. Rinse the filter once with PBS.
11. React the filter with the 2nd antibody (HRP-goat anti-mouse
IgG) dissolved in PBS containing 5% BSA, 100 /lg/ml yeast
tRNA and 100 /lg/ml salmon sperm DNA, for Ihr at RT.
12. Repeat steps 9 and 10
13. Immerse the filter in the DAB/HzOzlCo/Ni solution for 10
mins at RT (Adams 1981).
14. Wash the filter at least 3 times with DDW for 10 mins each
and dry up.
161
Immunohistochemical detection
1. Cut lx4 cm strips of nitrocellulose filters and immerse in
DDW for 30 mins at RT.
2. Replace DDW for 20x SSC and keep them for 30 mins at RT.
3. Place the filters on a sheet of Parafilm and dry up.
4. Apply 2 /ll of DNA solution containing DNA at various quantities per dot (10 ng,1 ng,100 pg, 10 pg, 1 pg).
5. Dry up completely (overnight air-dry is recommended).
6. Bake the filter at 80°C for 2 hrs exactly in an oven.
7. Place the filter inside a vinyl bag and incubate with a blocking solution consisting of 5% BSA (98-99% pure), 500 /lg/ml
normal goat IgG, 100 /lg/ml yeast tRNA, 100 /lg/ml salmon
sperm DNA, (0.05% NaN3) and PBS, for Ihr at RT.
8. Take the filter out of bag and replace the blocking solution
with the 1st antibody (mouse anti-T -T IgG; 1:80) dissolved in
PBS containing 5% BSA, 100 /lg/ml yeast tRNA, 100 /lg/ml
salmon sperm DNA and 0.05% NaN3, for 3 hrs at RT in a
vinyl bag.
9. Wash the filter with 0.075% Brij 35 in PBS at RT (4x 15 mins).
10. Rinse the filter once with PBS.
11. React the filter with the 2nd antibody (HRP-goat anti-mouse
IgG) dissolved in PBS containing 5% BSA, 100 /lg/ml yeast
tRNA and 100 /lg/ml salmon sperm DNA, for Ihr at RT.
12. Repeat steps 9 and 10
13. Immerse the filter in the DAB/HzOzlCo/Ni solution for 10
mins at RT (Adams 1981).
14. Wash the filter at least 3 times with DDW for 10 mins each
and dry up.