Ab5320

Heart tissue was pulverized in a glass douncer in NP40 lysis buffer (50 mM Tris [pH 8.0], 150 mM NaCl, 2 mM EDTA, 10 mM NaF, 10% glycerol, and 1% NP‐40) containing complete protease inhibitor cocktail (Roche), phosphatase inhibitor cocktails 2 and 3 (Sigma). Lysates sat on ice for 30 min with intermittent vortexing. Lysates were centrifuged (13k rpm, 10 min, 4°C). 50–100 μg of protein lysate was treated for 6–8 h with chondroitinase ABC (100 μU/mL; R&D Systems) before being resolved on 4%–12% bis‐tris gel (BioRad Criterion XT) by SDS/PAGE, transferred to nitrocellulose membrane (GE Life Sciences), and blocked in 5% nonfat milk (Blake, Parrish, et al., 2022 (link)). Samples were probed with mouse anti‐chondroitin‐4‐sulfate (1:1000; Millipore MAB2030), mouse anti‐chondroitin‐6‐sulfate (1:1000; Millipore MAB2035), rabbit anti‐NG2/CSPG4 (1:1000; Millipore AB5320), or rabbit anti‐CHST15 (1:1000; Proteintech 14298‐1‐AP). Blots were washed, incubated with goat anti‐mouse or anti‐rabbit HRP (1:10,000; Thermo 32430 & 32460, respectively), and bound antibody detected by chemiluminescence (Thermo). Protein expression was quantified with ImageJ densitometry and normalized to total protein as measured by Ponceau staining.

The slides containing the sections were blocked for 1 hr at room temperature in blocking buffer containing antibody buffer (100 mM L-lysine and 0.3% Triton X-100 in PBS) supplemented with 10% heat-inactivated normal goat serum. Primary antibodies diluted in antibody buffer with 5% goat serum were incubated overnight at 4°C. The next day, slides were washed 3 × 5 min with PBS with 0.2% Triton X-100 and secondary antibodies conjugated to Alexa Fluor (Molecular Probes) were applied for 2 hr at room temperature. Slides were mounted with the SlowFade Gold with DAPI mounting media (LifeTech #S36939), covered with 1.5 glass coverslip (Fisher #12544E) and sealed with clear nail polish. The following antibodies were used: Chk anti-GFP (Millipore #06-896, 1:500), Rb anti-SOX9 (Abcam #ab185966, 1:2000), Rb anti-ALDH1L1 (Abcam #ab-87117, 1:500), Rb anti-HA (CST #3724), Rb anti-S100β (Abcam #ab52642, 1:100), Ms anti-NEUN (Millipore #MAB377 1:100), Rb anti-NG2 (Millipore # Ab5320), Rb anti-MOG (Proteintech # 12690-1-ap), Rb anti-IBA1 (Wako #016-20001), Gp anti-VGLUT1 (Millipore #AB5905, 1:2000), Gp anti-VGLUT2 (Millipore #AB2251 1:3000, 1:5000), Rb anti-GLUA1 (Millipore #AB1504, 1:400), Rb anti-GLUA2 (Millipore #AB1768-I, 1:400), and Ms anti-Bassoon (Enzo #VAMP500, 1:500). All secondary antibodies were applied at 1:500 dilution.
The following mouse lines and antibody combinations were used: