Rapid bright-field detection of oligonucleotide primed in situ (PRINS)-labeled DNA in chromosome preparations and frozen tissue sections.
We describe a new application of a bright-field microscopic procedure for rapid enzyme cytochemical detection of repeated DNA sequences in metaphase preparations and frozen tissue sections. Various chromosome-specific oligonucleotide primers were used in up to three sequential primed in situ (PRINS) labeling reactions together with Taq DNA polymerase and biotin, digoxigenin and/or fluorescein isothiocyanate (FITC)-modified nucleotides. DNA target sequences were localized simultaneously by the precipitates of the horseradish peroxidase-diaminobenzidine (PO-DAB, brown color), alkaline phosphatase-Fast Red (APase-Fast Red, red color) and horseradish peroxidase-teramethylbenzidine (PO-TMB, green color) reaction in hematoxylin counterstained metaphases and interphase nuclei using a standard bright-field microscope. In addition, a protocol is reported for the application of PRINS to frozen tissue sections from normal colon and bladder epithelium. Methanol/acetic acid fixation in combination with a pepsin digestion before performing the PRINS reaction proved to be critical steps in the total procedure that permits access of the PRINS reactants, while preserving the morphology of the nuclei in the tissue. Quantification of PRINS signals showed the majority of epithelial cells with the expected two chromosome copies. The described procedures can be considered valuable tools for application in molecular cytogenetics, cell biology and pathology.
Speel
E J
EJ
Dept. of Molecular Cell Biology & Genetics, University of Limburg, Maastricht, Netherlands. ernstjan.speel@molcelb.rulimburg.nl
Lawson
D
D
Ramaekers
F C
FC
Gosden
J R
JR
Hopman
A H
AH
D001710:Biotin; D002875:Chromosomes / Q000737:chemistry*; D003106:Colon / Q000737:chemistry; D004247:DNA / Q000032:analysis*; D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D003854:Deoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D004076:Digoxigenin; D004848:Epithelium / Q000737:chemistry; D005456:Fluorescent Dyes; D006735:Horseradish Peroxidase; D006801:Humans; D017403:In Situ Hybridization / Q000379:methods*; D008214:Lymphocytes / Q000737:chemistry; D008297:Male; D008856:Microscopy, Fluorescence; D012091:Repetitive Sequences, Nucleic Acid; D001743:Urinary Bladder / Q000737:chemistry
D017931:DNA Primers; D003854:Deoxyribonucleotides; D005456:Fluorescent Dyes; D001710:Biotin; D004247:DNA; D006735:Horseradish Peroxidase; D004259:DNA-Directed DNA Polymerase; D004076:Digoxigenin