470
TORN KOCH
2. After incubation in stop buffer (step 4), immediately dehydrate the
slide in a -20°C ethanol series (70%, 90% and 99%, 3 min each).
3. Remove the slide from the 99% ethanol, drain and air-dry.
4. Prepare and mix a new reaction mixture as before, but using a different
type of labeled dNTP (in this case, e.g. fluorescein-dUTP (Roche».
5. Annealing and chain elongation: The slide should NOT be denatured
prior to the second PRINS, but is preheated to 55-65°C for 1 min. Then
the likewise preheated reaction mixture is put on the slide and spread
with a coverslip. Incubation is as above.
6. Place the slide in preheated stop buffer for 1 min at the temperature
used in step 5.
7. Wash the slide for 3 min at room temperature in washing buffer.
8. The slide is now ready for antibody staining and counterstainingl
mounting.
9. If a third PRINS reaction is desired, the wash (step 7) is omitted. The
slide is instead immediately dehydrated in ethanol once more to repeat
steps 2-8.
PRINS-FISH is done by adding the denatured FISH probe to the slide instead of the second PRINS probe (step 5). The usual FISH procedures can
be employed except that the slide should not be (re)denatured after the
PRINS, as this may cause the PRINS signal to disappear. Also, it is generally not necessary to do any proteinase treatments prior to PRINS-FISH.
We use PRINS-FISH in PRINS-painting (Hindkjrer et al. 1995) and PRINSCGH formats to combine painting data with locus specific data (unpublished).
Visualization of digoxigenin-Iabeled DNA in situ
1. Apply 50 Jll of fluorochrome conjugated anti-digoxigenin in blocking
solution (2 ng/Jll) to the slide. Incubate under a coverslip for 30-60 min
in the dark.
2. Wash 2x 5 min in 50 ml of washing buffer. The slide is now ready for
counterstaining and mounting.
TORN KOCH
2. After incubation in stop buffer (step 4), immediately dehydrate the
slide in a -20°C ethanol series (70%, 90% and 99%, 3 min each).
3. Remove the slide from the 99% ethanol, drain and air-dry.
4. Prepare and mix a new reaction mixture as before, but using a different
type of labeled dNTP (in this case, e.g. fluorescein-dUTP (Roche».
5. Annealing and chain elongation: The slide should NOT be denatured
prior to the second PRINS, but is preheated to 55-65°C for 1 min. Then
the likewise preheated reaction mixture is put on the slide and spread
with a coverslip. Incubation is as above.
6. Place the slide in preheated stop buffer for 1 min at the temperature
used in step 5.
7. Wash the slide for 3 min at room temperature in washing buffer.
8. The slide is now ready for antibody staining and counterstainingl
mounting.
9. If a third PRINS reaction is desired, the wash (step 7) is omitted. The
slide is instead immediately dehydrated in ethanol once more to repeat
steps 2-8.
PRINS-FISH is done by adding the denatured FISH probe to the slide instead of the second PRINS probe (step 5). The usual FISH procedures can
be employed except that the slide should not be (re)denatured after the
PRINS, as this may cause the PRINS signal to disappear. Also, it is generally not necessary to do any proteinase treatments prior to PRINS-FISH.
We use PRINS-FISH in PRINS-painting (Hindkjrer et al. 1995) and PRINSCGH formats to combine painting data with locus specific data (unpublished).
Visualization of digoxigenin-Iabeled DNA in situ
1. Apply 50 Jll of fluorochrome conjugated anti-digoxigenin in blocking
solution (2 ng/Jll) to the slide. Incubate under a coverslip for 30-60 min
in the dark.
2. Wash 2x 5 min in 50 ml of washing buffer. The slide is now ready for
counterstaining and mounting.