Hoechst reagent 33258

Indirect immunofluorescence assays were performed on UM cell lines (T97, T108, T142 and T143) grown on glass coverslips and acetone-fixed (10 min at −20 °C). Cells were incubated for 45 min with a primary antibody directed against the HTR2B protein (SAB4501476, Sigma-Aldrich, Oakville, ON, Canada) and used at an optimal dilution of 1:100 in phosphate-buffered saline (137 mM NaCl, 2.7 mM KCl, 6.5 mM Na₂HPO₄ and 1.5 mM KH₂PO₄) containing 1% bovine serum albumin. The secondary antibody (rabbit anti-mouse IgG (H + L) conjugated with Alexa-fluor^® 488 (1:400; Molecular Probes, Burlington, ON, Canada)) was incubated for 30 min. Cell nuclei were revealed following Hoechst reagent 33258 labeling (1:100; Sigma-Aldrich). They were observed with an epifluorescence microscope (Zeiss Imager.Z2; Zeiss Canada Ltd., North York, ON, Canada) and photographed (AxioVision software, Carl Zeiss Microscopy, Peabody, MA, USA).

hTECs were incubated with fresh DH medium containing 10 μM 5-bromo-2’-deoxyuridine (BrdU; Sigma Chemicals, St-Louis, MO) for 7 days (hTECs were fed fresh medium supplemented with 10 μM BrdU every 48 h). hTECs were then chased for 0, 7, 14 and 21 days by switching to BrdU free medium. Detection of both BrdU and ΔNp63α was conducted by immunofluorescence analyses performed as described [53 (link)] on 10-µm thick cryosections fixed for 10 min. at room temperature with 1% formol and 10 min. at −20 °C with methanol and in NaOH (0.07 N) for 10 min. at room temperature. The following primary antibodies were used: anti-human ΔNp63α (clone 250, 1:2000; rabbit polyclonal produced by MÉDIMABS, Montréal, Qc, Canada (not commercially available)) conjugated to cyanine 3, and a mouse monoclonal anti-BrdU antibody (347580 (monoclonal), 1:500; BD Pharmingen). Rabbit anti-mouse IgG H+L antibodies conjugated with Alexa 488 (A11059, 1:500; Invitrogen) and goat anti-rabbit IgG H+L antibodies conjugated with Alexa 594 (A11012, 1:1000; Invitrogen) were used as secondary antibodies. Cell nuclei were counterstained with Hoechst reagent 33,258 (1:100; Sigma Chemicals). Negligible background was observed for controls (primary antibodies omitted). Fluorescence was observed using a confocal microscope (Zeiss Imager. Z2 LSM 800; Zeiss Canada Ltd., North York, ON, Canada).

Cellular uptake of exosomes by hCECs and hCFs was assessed by confocal microscopy. Exosome were labeled with DiI fluorescent dye (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate; Thermo Fisher Scientific), a lipophilic membrane stain. Briefly, exosomes’ suspensions (100 μL each) were incubated with DiI dye for 20 min at 37 °C in the dark. Then labeled-exosomes were washed, collected and added to hCECs and hCFs cultures. Cells were incubated with labeled exosomes for 24 h prior to fixation in 4% formaldehyde. Prior to immunodetection, cells were permeabilized with 0.2% Triton X-100 for 10 min. Cell nuclei and actin filaments were counterstained with Hoechst reagent 33258 (1:100; Sigma) and phalloidin-Alexa 488 (1:200, Invitrogen, Carlsbad, CA, USA) respectively. Photos were taken with a confocal microscope (LSM 800; Zeiss, Toronto, ON, Canada).

Cells (1 × 10⁴/well) were fixed with 3.7% formaldehyde (Sigma−Aldrich) for 15 min and permeabilized with 0.2% Triton X-100 for 15 min. Subsequently, cells were blocked with 5% fetal bovine serum in phosphate-buffered saline (PBS; AMRESCO, Solon, OH, USA) for 1 h and then incubated with primary antibodies (1:1000 dilution, 1 h) targeting γ-H2AX, PARP1, p-p53^ser15, and p-ATM (Santa Cruz Biotechnology, Inc.). Cells were then incubated with secondary antibodies (1:1000 dilution, 1 h) conjugated with Alexa Fluor 488 or 546 (Invitrogen, Carlsbad, CA, USA). Nuclei were stained with Hoechst 33258 reagent (5 μM; Sigma−Aldrich) for 15 min. Stained cells were imaged using confocal microscopy (LSM-700, Carl Zeiss Microimaging, Oberkochen, Germany) and analyzed using Zen 2009 software.