A11059

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).

The 3 types of BAMs (mouse GFP-labeled C2C12, human BAM-M and human BAM-MM) and the human muscle strips were washed (3 × 5 min in PBS) and then removed from the attachment sites. Then, they were pinned on Styrofoam to preserve their original shape during fixation in 4% formaldehyde for 1 h and stored at 4 °C in PBS. Immediately before whole-mount staining, samples were fixed a second time in −20 °C methanol for 10 minutes and permeabilized in blocking buffer for 3 hours. Subsequently, the human BAMs and muscle strip were incubated overnight at 4 °C with a monoclonal mouse antibody against tropomyosin (Sigma, T9283, 1:100 in blocking buffer) and MF20 (DSHB), recognizing all myosin heavy chain (MHC) isoforms. Next, the human BAMs and muscle strips were washed and incubated with a polyclonal goat anti-mouse secondary antibody (Alexa Fluor 633, A-11059 or A-21063, Invitrogen, 1:200) for 30 minutes in the dark, followed by incubation with DAPI (Life Technologies, 0.1 µg/ml in PBS) for 1 hour. Samples were stored in PBS in the dark until visualized.

DNA combing was performed essentially as described in (Vannier et al., 2013 (link)). Briefly, Rtel1^f/fTerc^+/+ and Rtel1^f/fTerc^-/ MAFs were infected with control- or Cre-expressing adenovirus. Cells were pulse-labeled with IdU/CldU for 20 minutes, each pulse. DNA fibers were extracted in agarose plugs and stretched on silanized coverslips with the molecular combing system (Genomic Vision). CldU was detected with rat anti-BrdU antibody (BU1/75, AbCys), followed by goat anti-rat coupled to Alexa 594 (A11007, Molecular Probes) and finally by chicken anti-goat coupled to Alexa 594 (A21468, Molecular Probes). IdU was detected with Mouse anti-BrdU coupled to FITC antibody (BD44, Becton Dickinson), followed by rabbit anti-mouse coupled to Alexa 488 (A11059, Molecular Probes) and finally by donkey anti-rabbit coupled to Alexa 488 (A21206, Molecular Probes). DNA fibers were captured with a Zeiss Axio Imager M1 microscope equipped with an ORCA-ER camera (Hamamatsu) controlled by Volocity 6.3 software (Improvision).

Cells were rinsed twice with PBS and treated with ice-cold pre-extraction buffer (pH 6.8) containing 10 mM HEPES, 100 mM NaCl, 300 mM sucrose, 1 mM magnesium chloride, 1 mM EGTA, 1 mM dithiothreitol, 1 mM PMSF, 10 μg ml⁻¹ aprotinin and 0.5% Triton X-100 for 2 min. The cells were then fixed with ice-cold pure methanol for 30 min and then incubated with blocking buffer (10% FBS and 0.2% Triton X in PBS) for 30 min. Primary antibodies against cytokeratin AE1/AE3 (1:100; Dako, #M3515), Zta (1:1000; Argene, #11-007), EA-D (1:500, a gift from Professor Jaap Middeldorp) and BALF2 (1:500) (a kind gift from Professor Tatsuya Tsurumi, Aichi Cancer Center Research Institute, Japan) were used. Alexa Fluor-conjugated secondary antibodies (1:500) were from Molecular Probes (#A11059 for cytokeratin AE1/AE3, Zta and EA-D and #A21206 for BALF2). The percentage of positive cells was quantified by calculating 10 microscopic views with over 2000 cells included.

DNA combing was performed essentially as described in (Vannier et al., 2013 (link)). Briefly, Rtel1^f/fMsh2^+/+ and Rtel1^f/fMsh2^-/ MEFs were infected with control- or Cre-expressing adenovirus. Cells were pulse-labeled with IdU and CldU for 20 minutes each. Cells were then collected and the DNA was extracted according to DNA extraction kit provided by Genomic Vision. DNA fibers were extracted in agarose plugs and stretched on silanized coverslips with the molecular combing system (Genomic Vision). CldU was detected with rat anti-BrdU antibody (BU1/75, AbCys), followed by goat anti-rat coupled to Alexa 594 (A11007, Molecular Probes) and finally by chicken anti-goat coupled to Alexa 594 (A21468, Molecular Probes). IdU was detected with Mouse anti-BrdU coupled to FITC antibody (BD44, Becton Dickinson), followed by rabbit anti-mouse coupled to Alexa 488 (A11059, Molecular Probes) and finally by donkey anti-rabbit coupled to Alexa 488 (A21206, Molecular Probes). DNA fibers were captured with a Zeiss Axio Imager M1 microscope equipped with an ORCA-ER camera (Hamamatsu) controlled by Volocity 6.3 software (Improvision).

After treatments, cells were loaded with Mito tracker (Cayman Chemical, Ann Arbor, MI, USA) in PBS for 30 min at 37 °C, washed with PBS and then the cells were fixed with methanol for 10 min at −20 °C and blocking in 5% BSA in PBST for 30 min. Fixed cells were incubated overnight at 4 °C with primary antibody nephrin (ab216341, Abcam, Cambridge, MA, USA), cytochrome c (12963S, Cell Signaling, Beverly, MA) or TRPC6 (ab62461, Abcam, Cambridge, MA, USA) diluted in PBST and then washed three times in PBST and incubated for 1 h at room temperature with anti-rabbit or anti-mouse Alexa Fluor 488 or 594 (A-11059 and A27027, Molecular Probes). The mitochondria were labeled with Mito tracker and the nuclei were visualized by DAPI (ab228549, Abcam, Cambridge, MA, USA) staining. Images were taken using LEICA laser scan microscope.