Sc 17320

hMSCs and mMSCs were fixed with formaldehyde (4% in PBS), permeabilized with Triton X-100 (0.4% in PBS), incubated with blocking buffer (10% donkey serum in PBS), and stained with primary antibody overnight at 4 °C. Then, cells were treated with secondary antibodies for 1 h at room temperature. Hoechst 33342 (Invitrogen) was used for nuclear DNA staining. The antibodies used are as follows: anti-RANK (ab12008, 1:100), anti-CD90 (Abcam, ab21624, 1:200), anti-DMP1 (Santa Cruz, sc-17320, 1:100).

Immunocytofluorescence analysis was performed as described previously^{68 (link)}. Cells were fixed with 4% paraformaldehyde in phosphate buffered-saline (PBS), permeabilized for 10 min with 0.2% Triton X‐100 in PBS, exposed for 30 min to 3% bovine serum albumin in PBS (blocking solution), incubated first overnight at 4 °C with primary antibodies diluted in the blocking solution and then for 1 h at room temperature with Alexa Fluor–labeled secondary antibodies (1:1000 dilution) (A21206, A21202, A11039, A31572, A31570, A31571, A31573, A32795, A21447, Molecular Probes) and Hoechst 33342 (1:1000 dilution) (Molecular Probes) in blocking solution, and then mounted in Mowiol (Calbiochem). Primary antibodies and their dilutions were as follows: anti–acetylated α-tubulin, 1:500 (T6793, Sigma-Aldrich); anti-Id1, 1:500 (BCH-1/37-2, Biocheck); anti-β-catenin, 1:200 (C2206, Sigma-Aldrich); anti-Foxj1, 1:500 (14-9965, eBioscience); anti-Sox2, 1:200 (sc-17320, Santa Cruz); anti-S100β, 1:200 (S2657-0.2ML, Sigma-Aldrich); anti-BrdU, 1:500 (ab6326, Abcam); anti-cleaved caspase3, 1:500 (9664S, Cell Signaling Technology); and anti-GFAP, 1:1000 (ab4674, Abcam). Images were acquired with a laser‐scanning confocal microscope (TSC‐SP5, Leica or LSM 880, Zeiss) and processed with ImageJ software (NIH).

Retinal explant and transgenic mouse (below) tissue was fixed in 2% paraformaldehyde for 30 to 120 minutes at room temperature, cryopreserved through 30% sucrose, and frozen in OCT (Sakura Finetech, Torrance, CA, USA). Cryosections were cut at 10 μm and immunostained as previously described [22 (link), 23 (link)]. The following primary antibodies were used: rat anti-Blimp1 (1:100) (sc47732, Santa Cruz, Dallas, TX, USA); chicken anti-GFP (1:1000) (ab13970, Abcam, Cambridge, MA, USA); goat anti-Otx2 (2.5μg/mL) (BAF1979, R&D Systems, San Jose, CA, USA); rabbit anti-Pax6 (1:500) (901301, BioLegend, San Diego, CA, USA); guinea pig anti-Ptf1a (1:500) (a gift from Jane Johnson, UTSW); rabbit anti-red fluorescent protein (1:500) (ab34771, Abcam); goat anti-Sox2 (1:100) (sc17320, Santa Cruz); and sheep anti-Vsx2 (1:200) (X1179P, Exalpha, Shirley, MA, USA). Three to five z-stack images were collected using an Olympus FV1000 (Waltham, MA, USA) or Nikon C2 (Melville, NY, USA) laser scanning confocal microscope. Maximum intensity projection images were generated with ImageJ [26 (link)] and minimally processed with Adobe Photoshop (San Diego, CA, USA).

Immunostaining was performed according to our previously described^{9 (link)}. An antibody used to detect γH2A.X (1:100; #2577) was purchased from Cell Signaling Technology (Beverly, MA). Antibodies used to detect octamer-binding transcription factor 4 (OCT4) (1:100; sc-8628) and SOX2 (1:100; sc-17320) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-H3K9ac antibody (1:500; ab10812) was purchased from Abcam (Cambridge, MA). The embryos were examined under a confocal laser scanning microscope (Zeiss LSM 510 and 710 META; Zeiss, Oberkochen, Germany). Fluorescence intensities were quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA)^{68 (link)}. The number of γH2A.X foci was quantified using the Zeiss software, and foci larger than 0.3 μm³ were deemed to be sites of DSB^{25 (link), 65 (link)}.