Phosphate buffered detergent

Cells grown on coverslips were fixed in cold methanol-acetic acid (3:1) for 3 min and 4% paraformaldehyde–PBS for 20 min. Immunofluorescence was performed as described above. Antibodies were fixed in situ with methanol-acetic acid (3:1) at room temperature for 10 min and 2% paraformaldehyde–PBS at room temperature for 2 min. Cells were treated with RNase A and dehydrated in a 70%, 90%, and 100% ethanol series for 3 min each. Full-length HPV16 DNA was fluorescently labeled using the Ulysis nucleic acid labeling kit (Life Technologies). Seventy-five to 360 ng labeled fluorescence in situ hybridization (FISH) probe in hybridization buffer (Empire Genomics) was added to the coverslip, and DNA was denatured at 75°C for 5 min, followed by hybridization at 37°C overnight. Cells were washed with 1× phosphate-buffered detergent (Qbiogene) for 5 min at room temperature, 1× wash buffer (0.5× SSC, 0.1% SDS) for 5 min at 65°C, and 1× phosphate-buffered detergent (Qbiogene) for 5 min at room temperature. Coverslips were mounted in ProLong Gold containing DAPI for analysis by confocal microscopy.

Cells grown on coverslips were fixed in cold methanol-acetic acid (3:1) for 1–3 minutes followed by 4% paraformaldehyde–PBS for 10 minutes. Cells were treated with RNase A for 1 hour at 37°C and dehydrated in a 70%, 90%, and 100% ethanol series for 3 minutes each. Green 5-Fluorescein dUTP labelled BAC clone RP11-1140H22 (chr2: 28,504,596–28,660,966; hg19) was purchased from Empire Genomics. DNA-FISH probe against full-length HPV16 DNA was fluorescently labeled using the Alexa Fluor-594 FISH Tag DNA Multicolor Kit (Life Technologies) following manufacturer’s protocol. To each coverslip, 40–50 ng labeled DNA FISH-probe in hybridization buffer (Empire Genomics) supplemented with 50 μg/ml human Cot-1 DNA (Invitrogen) was added. DNA denaturation was performed at 75°C for 5 minutes, followed by hybridization at 37°C overnight. Cells were washed for 5 minutes each with 1x phosphate-buffered detergent (Qbiogene) at room temperature, 1x wash buffer (0.5x SSC, 0.1% SDS) at 65°C, and 1x phosphate-buffered detergent at room temperature. Coverslips were mounted in ProLong Gold (ThermoFisher) containing DAPI for analysis by confocal microscopy.