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

454 Speel, Ramaekers, and Hopman
of nucleic acid targets, lack of crossreaction between the different protein and nucleic
acid detection procedures, and good color contrast and stability of the fluorochromes
and enzyme cytochemical precipitates applied. A variety of procedures have been
reported that combine ICC and ISH (for a review, see ref. 10,12), in most cases
applying ICC before ISH to prevent the destruction of antigenic determinants by
the ISH procedure because of enzymatic digestion, postfixation, denaturation at
high temperatures, and hybridization in formamide. For reasons of rapidity, probe
accessibility, and lack of formamide for hybridization, the substitution of ISH by
PRINS may be an extra advantage in such a combined procedure.
Here, we present three protocols for combined ICC and PRINS DNA labeling. In the
first procedure, a sensitive, high-resolution fluorescence alkaline phosphatase (APase)-
Fast Red ICC staining method (13,14) is performed before subsequent PRINS labeling
of DNA target sequences to enable the simultaneous detection of surface antigens
(EGF receptor, neural cell adhesion molecule) and repeated chromosome-specific DNA
sequences in somatic cell hybrid and tumor cell lines. The fact that the Apase-Fast Red
precipitate withstand subsequent proteolytic digestion and denaturation steps guarantees
the most optimal conditions for efficient PRINS labeling (15). The second procedure
has been recently described to investigate chromosome distribution and segregation in
cells during processes, such as polyploidization and aneuploidization. This protocol
starts with the PRINS labeling of chromosome centromeres followed by the staining
of the mitotic spindle by tubulin ICC, because the authors found the antibody epitope
to be better preserved when ICC came after DNA labeling (16,17). The third protocol
has been used on metaphase chromosomes to identify possible relationships of different
families of DNA sequences with, for example, proteins associated with different
chromosome-specific structures, such as the kinetochore complex. This approach again
starts with ICC staining followed by PRINS DNA labeling (18,19).
2. Materials
2.1. Protocol 1
2.1.1. Fluorescence Immunophenotyping by Alkaline Phosphatase Cytochemistry
1. Cold methanol (–20°C), cold acetone (4 and –20°C), and cold 70% ethanol (–20°C).
2. Normal goat serum (NGS).
3. Monoclonal antibody EGFR1, directed against the epidermal growth factor receptor (a
kind gift of V. van Heyningen, Edinburgh, UK).
4. Monoclonal antibody 163A5, directed against a cell-surface marker of J1-C14 cells (20).
5. Monoclonal antibody RNL1, directed against the neural cell adhesion molecule
(N-CAM) (21).
6. Alkaline phosphatase-conjugated goat anti-mouse IgG (GAMAPase) (Dako, Glostrup,
Denmark).
7. Naphthol-ASMX-phosphate (Sigma, St. Louis, MO).
8. Fast Red TR (Sigma).
9. Polyvinylalcohol (PVA), MW 40,000 (Sigma).
10. 10× phosphate-buffered saline (PBS): 1.37 M NaCl, 30 mM KH 2 PO 4 , 130 mM Na 2 HPO 4 .
11. APase buffer: 0.2 M Tris-HCl, pH 8.5, 10 mM MgCl 2 , 5% PVA. Dilute from stock solutions
1 M Tris-HCl, pH 8.5 and 1 M MgCl 2 and add 5% (w/v) PVA. Dissolve PVA by using
a microwave.
12. Blocking buffer: 1× PBS (diluted from stock 10× PBS), 0.05% Triton X-100, 2–5% NGS.