Alexa 488 or alexa 595 labeled anti species specific antibody conjugates

The antibodies used and relevant dilutions are as follows: SAMHD1, 1:1,000 (Cell Signaling Technology); BrdU, 1:200 (Cell Signaling Technology); cyclin E, 1:1,000 (Sigma-Aldrich); and V5, 1:500 (Abcam). Immune complexes were visualized using Alexa 488- or Alexa 595-labeled anti-species-specific antibody conjugates (Molecular Probes). Cellular DNA was stained with 4′,6-diamidino-2-phenylindole (DAPI; Santa Cruz sc-3598). Fluorescent in situ hybridization (FISH) staining for HPV16 genomes was performed using digoxigenin (DIG)-labeled HPV16 genomes, as described previously (48 (link), 49 (link)). Immunofluorescence was quantified, using a Vectra Polaris automated imaging system, whereby whole stained sections were scanned computationally and the intensity calculated compared to a negative background control (secondary antibody only) and a positive localization control (DAPI). The same imaging parameters were used for each slide. For each sample, two sections from three individual rafts were scanned to generate average values. Immunofluorescence was observed using a LSM 710 laser scanning microscope and Zen 2011 software (Carl Zeiss). Images were assembled in Adobe Photoshop CS 6.0.

Cells were grown on coverslips to 70% confluence, fixed with formaldehyde, and permeabilized with NP-40. Cells were incubated with the primary antibody for 1 h (phospho-yH2.AX; Cell Signaling Technology 20E3; 1/500), diluted in 10% normal goat serum. Coverslips were washed three times in PBS-Tween (0.1% Tween), and immune complexes were visualized using Alexa 488- or Alexa 595-labeled anti-species-specific antibody conjugates (Molecular Probes). Cellular DNA was stained by inclusion of 4′,6-diamidino-2-phenylindole (DAPI; Santa Cruz sc-3598) in the penultimate wash. Microscopy was performed at the VCU Microscopy Facility. Immunofluorescence was observed using an LSM 700 laser scanning microscope and ZEN 2011 software (Carl Zeiss). Images were assembled in Adobe Photoshop CS 6.0.