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

456 Speel, Ramaekers, and Hopman
Fig. 1. Fluorescence detection of (A) chromosome 9 centromeres with digoxigenin/
SHADigFITC in T24 cells after PRINS and Vectashield embedding with PI counterstaining. (B)
chromosome 7 and 9 centromeres with, respectively, biotin/AvidinTexasRed anf digoxigenin/
SHADigFITC after double-target PRINS and Vectashield embedding with DAPI counterstaining.
(C) EGF receptor with alkaline phosphatase-Fast Red and four chromosome 7 centromeres
with biotin/avidinFITC in C121-TN6 cells after immunocytochemistry followed by PRINS
and Vectashield embedding. (D) NCAM antigen with alkaline phosphatase-Fast Red and three
chromosome 9 centromeres with digoxigenein/SHADigFITC in H460 cells after immunocytochemistry
followed by PRINS and Vectashield embedding.
30. Bio–Rad MRC 600 confocal scanning laser microsope equipped with the laser lines 488,
568, and 648 nm (Bio–Rad Laboratories, Veenendaal, The Netherlands).
2.2. Protocol 2
2.2.1. PRINS DNA Labeling
1. Dried skimmed milk powder.
2. PRINS reaction mixture components: dNTPs, labeled dUTPs, oligonucleotide primers, and
Taq DNA polymerases as described in Subheading 2.1.2., items 2–6 and 16.
3. 1× PBS (diluted from 10× PBS, see Subheading 2.1.1., ref. 10) containing 0.1% EDTA
and 0.2% BSA.
4. Methanol: acetic acid (91), freshly prepared.
5. 1× PBS (diluted from 10× PBS, see Subheading 2.1.1., ref. 10).
6. Permeabilization buffer: 1× PBS containing 0.1% Triton X 100.
7. A series of 70, 96, and 100% ethanol.
8. Denaturation buffer: 70% formamide/2 × SSC pH 7.0.