Rabbit anti mouse ng2

OCT embedded sections (14 µm thick) were thawed and fixed with cold acetone for 10 min. Sections were then washed with PBS and incubated in blocking buffer (1% goat serum (Jackson Immuno Research Labs, West Grove, PA) + 0.05% Tween-20 in PBS) for 1 h at RT before being incubated in primary antibody solution (1 : 100 rat anti-mouse CD31 (BD Pharmingen, San Diego, CA) and 1 : 200 rabbit anti-mouse NG-2 (Millipore, Billerica, MA)) overnight at 4 °C. Sections were again washed with PBS and incubated in blocking buffer for 10 min at RT before being incubated for 2 h at RT in secondary antibody solution (1 : 100 goat anti-rat Alexa 568, and 1 : 100 goat anti-rabbit Alexa 488 (Invitrogen, Grand Island, NY)), and DAPI nuclear stain (1 : 400 dilution, Invitrogen, Grand Island, NY). Sections were then washed twice in PBS, mounted and imaged using a Nikon C2 confocal microscope. CD31-, NG2-, and DAPI-positive area fractions (%) reported were determined by normalizing the positively stained area to overall image area using ImageJ. All hematoxylin and eosin (H&E) staining of sections was conducted by the Translational Pathology Core Laboratory (TPCL) at UCLA. For each condition (n = 4), five images were quantified over three different sections, each ≥ 140 µm apart.

DR-ESCs were assessed for protein markers using immunocytochemistry and confocal microscopy. Cultures were fixed for 15 minutes at room temperature in 4% PFA, and immunostained as previously described^{37 (link),38 (link)}. Primary antibodies (Abs) used were: rat anti-mouse/human Oct3/4 at 1:200, rat anti-mouse platelet-endothelial cell adhesion molecule-1 (PECAM-1)/CD31 (BD Biosciences) at 1:1000, rabbit anti-mouse NG2 (Millipore) at 1:200, and rabbit polyclonal anti-phospho-Histone H3 (PH3, ser10, Millipore) at 1:500, rat anti-mouse PDGFRβ at 1:400, rabbit anti-mouse Desmin at 1:500, mouse anti-mouse A2B5 at 1:100. Secondary antibodies used were donkey anti-rat AlexaFluor647 (IgG; H+L) at ½ the primary Ab dilution factor (Invitrogen), and donkey anti-rabbit AlexaFluor647 (IgG; H+L) at ½ the primary Ab dilution. Incubation with DAPI for 30 min followed all staining. ESC cultures were imaged on a Zeiss LSM880 confocal microscope.