Anti fade gold

Monoclonal antibodies 33L1-7 and H16.56E, which recognize a linear highly conserved epitope (residues 303-313 of HPV16 L1) and a conformational HPV16 L1-specific epitope, respectively, were described previously. Rabbit polyclonal HPV18 antibody raised against HPV18 VLPs was generously provided by Neil D. Christensen, Hershey Medical Center, Hershey, PA, USA [54 (link),55 (link),56 (link)]. A rabbit polyclonal antibody against LN332 (ab14509) (Abcam, Cambridge, MA, USA) was used to detect ECM depositions. The β-actin antibody was purchased from Santa Cruz Biotechnologies (sc-47778) (Dallas, TX, USA). Alexa Fluor-labeled secondary antibodies and Gold Antifade were purchased from Life Technologies. Heparin was purchased from Iduron (Manchester, UK). Marimastat (444289), batimastat (196440) and GM6001 (364206) were obtained from EMD Biochemicals (Darmstadt, Germany). For real-time PCR, SYBR-green from BioRad (Hercules, CA, USA) (IQ™ supermix^® #170-8880) was used. A Click-iT EdU imaging kit and Alexa Fluor-Labeled antibodies were purchased from Invitrogen. Peroxidase-conjugated AffiniPure goat anti-mouse antibodies were purchased from Jackson ImmunoResearch (West Grove, PA, USA). Synthesis of DSTP27, an N,N'-bisheteryl derivative of Dispirotripiperazine (DSTP) has been described before [57 (link),58 (link)].

HFK cells were infected with EdU-labeled pseudovirions using ECM-to-cell transfer on glass slides. EdU staining was performed according to the manufacturer’s directions. In brief, at the indicated times post infection, cells were washed with PBS and fixed with 4% paraformaldehyde for 15 min at room temperature, washed, permeabilized with 0.5% Triton X-100 in PBS for 10 min, washed, and blocked with 5% goat serum in PBS for 30 min followed by a 30 min incubation with Click-iT reaction cocktail containing AlexaFluor 555 for EdU-labeled pesudogenome detection. After extensive washing, cells were incubated for 30 min with anti-PML (BETHYL; A301-167A), and anti-laminin A/C (Sigma; SAB4200236) primary antibodies at room temperature, washed again extensively, and subsequently incubated with AlexaFluor488- and AlexaFluor647-tagged secondary antibodies (Molecular Probes; A11029, A21245) for 30 min. After extensive washing with PBS, cells were mounted in ‘Gold Antifade’ containing DAPI (Life Technologies; P3693).

Cells were arrested in metaphase by treatment with colcemid for 4 h. Cells were then subsequently fixed and repeatedly washed in ice-cold methanol: acetic acid (3∶1), applied onto slides and embedded in DAPI mount (Anti-fade Gold, Invitrogen). Chromosome numbers in metaphase nuclei were assessed using a fluorescence microscope. Statistical significance values were calculated in Prism 5.0 software (GraphPad) using Student's t-test with Welch's correction.

DNA fiber analyses were carried out as described previously [33 (link)]. For measuring sister fork asymmetry, cells were labeled with CldU (33 μM) for 30 min followed by exposure to 2 mM HU for 2 h and chased with IdU (340 μM) for 40 min before harvesting in PBS. Cells were lysed (lysis buffer: 200 mM Tris-HCl (pH 7.4), 50 mM EDTA, 0.5% SDS) and DNA fibers stretched onto glass slides and fixed in methanol:acetic acid (3:1, Merck). After rehydration in PBS, these were denatured with 2.5 M HCl for 1 h, washed with PBS and blocked with 2% BSA in PBS containing 0.1% Tween 20 for 30 min. The newly replicated CldU and IdU tracks were immunostained using anti-BrdU primary and appropriate secondary antibodies. Coverslips were mounted using Antifade Gold (Invitrogen). Images were acquired on a Leica DMI 6000 fluorescence microscope and analyzed using Fiji software [34 (link)]. Statistics were calculated in Graph Pad Prism (GraphPad Software Inc., San Diego, CA, USA).

Cells were fixed in 4% PFA and washed with 1xPBS prior to staining. The cells were permeabilized using 0.2% Triton-X in 1xPBS (PBST) for 5 min. The cells were blocked using 10% FBS for 1 h. Primary antibodies were added and incubated at RT for 1-2 h. The cells were washed with 1xPBST. Secondary fluorescent antibodies were used at 1:500 dilution and incubated at RT for 45 min. The cells were washed with 1xPBS and mounted using anti-fade gold (Thermofisher Scientific).
Immunofluorescence analysis was performed on cryo-sections as described previously [39 (link)] from patient samples using citrate buffer (pH 6.0) for antigen retrieval by boiling for 20 min. The slides were cooled at RT for 30 min prior to permeabilization using 0.2% Triton-X 100 in 1xPBS. Following this, blocking was done in 10% FBS and primary antibodies were used at the requisite dilutions. The slides were then incubated overnight at 4 °C. After washes in 1xPBS, the secondary staining was done using secondary Alexa fluorophores and vectashield was used for mounting. Images were taken using the Zeiss 710 confocal microscope.

Tissue samples of the skin were fixed in 4% formaldehyde for 24 hours and then stored in 70% ethanol. The fixed samples were processed in a histomat (Leica Biosystems) and embedded in paraffin as part of routine procedures. The paraffin-embedded samples were cut into 3-μm sections.
For immunostaining, the sections were deparaffinized with xylene and ethanol. Antigen retrieval was performed using sodium citrate buffer in a water bath set at 58 °C. After overnight incubation, the slides were cooled in the fridge for 10–20 minutes. Subsequently, they were washed with Tris-buffered saline (TBS), and blocking buffer (1% BSA in 1 × TBS) was added for 60 minutes at room temperature.
After the removal of the blocking buffer, the first antibody was diluted in 100 μl of 1 × TBS with 1% BSA (at a dilution of 1:100) and incubated overnight at 4 °C with parafilm sealing the slides (Table 6 shows the antibodies used). After 2 washes with 1 × TBS, the second antibody was added at a concentration of 1:400 in 100 μl of blocking buffer and incubated for 60 minutes at room temperature.
The slides were then washed in 1 × TBS and sealed with cover slides with mounting medium (anti-fade gold, Thermo Fisher Scientific). Images of the stained sections were captured using an Axiokop 40 CFL camera (Zeiss). In Adobe Photoshop CC, tonal correction was applied to enhance contrasts within the images.