John M. S. Bartlett.pdf - Bio-Nica.info

PRINS and Immunocytochemistry 459
7. Place 40 µL of blocking buffer under a coverslip on the slide and leave for 5 min at room
temperature in a humid chamber to reduce background staining in the detection procedures.
8. Wash slides for 1 × 5 min in washing buffer in a Coplin jar.
9a. For reactions using Biotin-16-dUTP: Dilute AvFITC 1:100 in blocking buffer and apply
50 µL under a coverslip. Incubate slides for 30 min at 37°C in a humid chamber (see
Note 13).
9b. For reactions using Digoxigenin-11-dUTP: Dilute SHADigFITC 1100 in blocking buffer
and treat as in 9a (see Note 13).
9c. Fluorescein-12-dUTP needs no additional reporter and is simply mounted as described
in 11 (see Note 13).
10. Wash slides for 2 × 5 min in washing buffer in a Coplin jar. Optionally, you may wash the
slides for 5 min in 1× PBS and dehydrate them.
11. Mount the slides in Vectashield containing 0.5 µg/mLDAPI.
12. Examine the slides under a fluorescence microscope. Selected cells can be either directly
photographed using Kodak 400 ASA film, visualized with a charge-coupled device (CCD)
camera, or scanned with a confocal scanning laser microscope (CSLM).
3.2. Protocol 2
3.2.1 PRINS DNA Labeling
1. Hybrid and tumor cell lines are cultured on glass slides, as described in Subheading
3.1.1., step 1), or in suspension according to standard methods (16,17,20,22,23). Cells are
rinsed in 1× PBS/0.1% EDTA/0.2% BSA, cytocentrifuged on glass slides at 65g for 4 min
(in case of growth in suspension), fixed in methanolacetic acid (91) for 10 min at room
temperature in a Coplin jar, and airdried.
2. Rehydrate cells in 1× PBS for 5 min, permeabilize them with 0.1% Triton X 100 in
1× PBS for 5 to 10 min (both in a Coplin jar), dehydrate in a series of 70, 96, and 100%
ethanol and airdry.
3. Denature slides in 70% formamide/2 × SSC, pH 7.0, for 2 min at 70°C in a Coplin jar,
dehydrate in a series of 70, 96 (both at 4°C), and 100% ethanol, and airdry.
4. Prepare the PRINS reaction mix and apply it on the prewarmed (annealing temperature)
slides (on the block of the thermal cycler) as described in Subheading 3.1.2., steps 2
and 3).
5. Perform the PRINS reaction for 5 min at the appropriate annealing temperature (see Note 11)
and for 15 min at 72°C for chain elongation on a thermal cycler.
6. Stop the PRINS reaction by removing the coverslips (see Note 12) and wash the slides
in washing buffer for 3 × 5 min at room temperature followed by a 5-min wash step in
1× PBS (all steps in a Coplin jar).
7. Apply a blocking and wash step as described in Subheading 3.1.2., steps 7 and 8.
8. In case of PRINS labeling with Biotin-16-dUTP or Digoxigenin-11-dUTP, detect the
haptens as described in Subheading 3.1.2., steps 9 and 10).
3.2.2. Immunofluorescence Detection of the Mitotic Spindle
1. Incubate the slides for 30 to 45 min at room temperature with 50 µL of mouse
anti-β-tubulin primary antibody, diluted 150 in blocking buffer, under a coverslip in
a humid chamber.
2. Wash the slides for 2 × 5 min with washing buffer in a Coplin jar.
3. Incubate slides for 30 to 45 min at room temperature with 50 µL of DAMTRITC, diluted
1100 in blocking buffer (see Note 4), under a coverslip in a humid chamber.