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

458 Speel, Ramaekers, and Hopman
3. Methanol:acetic acid (31).
4. A series of cold (4°C) 70, 96, and 100% ethanol.
5. Denaturation solution: 30 mM NaOH/1 M NaCl (pH >12).
6. PRINS reaction, detection, and mounting components, as described in Subheading 2.1.2.,
items 2–26.
7. Slide evaluation equipment, as described in Subheading 2.1.2., items 27–30.
3. Methods
3.1. Protocol 1
3.1.1. Fluorescence Immunophenotyping by Alkaline Phosphatase Cytochemistry
1. Hybrid (C121-TN6, J1-C14) and tumor (H460) cell lines are cultured on glass slides
by standard methods (20,22,23), fixed in either cold methanol (–20°C) for 5 s and cold
acetone (4°C) for 3 × 5 s, cold acetone (–20°C) for 10 min, or 70% ethanol (–20°C) for
10 min, air-dried, and stored at –20°C until use (see Note 3).
2. Incubate slides for 10 min at room temperature with 100 µL of blocking buffer under a
coverslip in a humid chamber.
3. Remove the coverslip, discard the blocking buffer, and incubate the slides for 30 to 45 min
at room temperature with 50 µL of undiluted culture supernatant of the appropriate antigenspecific
monoclonal antibody containing 2% NGS under a coverslip in a humid chamber.
4. Wash slides for 2 × 5 min with washing buffer in a Coplin jar.
5. Incubate slides for 30 to 45 min at room temperature with 50 µL of GAMAPase, diluted
150 in blocking buffer (see Note 4), under a coverslip in a humid chamber.
6. Wash slides for 5 min with washing buffer, and for 5 min with 1× PBS.
7. Visualize the antigen with the alkaline phosphatase-Fast Red (APase-Fast Red) reaction:
Mix 4 mL of APase buffer, 1 mg of naphthol-ASMX-phosphate in 250 µL of buffer without
PVA and 5 mg of Fast Red TR in 750 µL of buffer without PVA just before use and overlay
each sample with 100 µL under a coverslip. Incubate the slides for 5 to 15 min at 37°C
and wash 3× 5 min with 1× PBS (see Notes 5–7).
3.1.2 PRINS DNA Labeling
1. Process cells for PRINS as follows: wash slides for 2 min at 37°C with 0.01 M HCl, incubate
the samples with 100 µg/mLpepsin in 0.01 M HCl for 20 min at 37°C, wash again with
0.01 M HCl for 2 min, and post-fix the slides in 1% formaldehyde in 1× PBS for 10 min
at room temperature. Wash cells in 1× PBS for 5 min at room temperature, followed by a
wash step in 1× Taq buffer for 5 min at room temperature (all steps in a Coplin jar).
2. Prepare the PRINS reaction mix on ice as follows: Dilute 100 mM dATP, dGTP, and dCTP
110 with distilled water. Dilute 100 mM dTTP 1:100. Put together in a microcentriphuge
tube: 1 µL of each of the diluted dNTPs, 1 µL of either 1mM Biotin-16-dUTP, Digoxigenin-
11-dUTP, or Fluorescein-12-dUTP (see Note 8), 5 µL of 10× Taq buffer, 250 ng of
oligonucleotide (see Note 9), 1 U Taq polymerase, and distilled water to 50 µL.
3. Place 40 µL of this mixture under a coverslip on the slide, seal with rubber cement, air-dry
the rubber cement, and transfer to the heating block of the thermal cycler.
4. Each PRINS reaction cycle consists of 2 min at 94°C (denaturation of cellular DNA, see
Note 10), 5 min at the appropriate annealing temperature (see Note 11) and 15 min at
72°C for in situ primer extension.
5. Stop the PRINS reaction by transferring the slides (after removal of the rubber solution
seal) to 50 mLof PRINS stop buffer in a Coplin jar at 65°C for 1 min (see Note 12).
6. Transfer the slides to washing buffer at room temperature and wash 5 min.