C8035

Tissue samples or cerebellar slices or purified OPC cells were homogenized with RIPA lysis buffer and protein concentration was determined by Pierce BCA protein assay kit according to the manufacture’s instructions (Thermo Fisher). Then, equal amounts of protein were loaded onto 15% SDS-PAGE gels, and electrophoretically transferred to PVDF membranes (Millipore). The membranes were blocked in 0.1% TPBS buffer with 5% non-fat milk for 1 h at room temperature and probed with indicated primary antibodies overnight at 4 °C and followed by secondary antibodies conjugated to horseradish peroxidase (HRP). The following primary antibodies were used: MBP (SMI-99P, Covance, 1:1000), CS56 (C8035, Sigma, 1:1000), Laminin (L9393, Sigma, 1:1000), GAPDH (AF5718, R and D Systems, 1:1000), MMP-2 (AF1488, R and D Systems, 1:1000), MMP-10 (MAB910, R and D Systems, 1:1000), and β-actin (sc-47778, Santa Cruz, 1:1000). Enhanced chemiluminescence was performed with a West Pico Kit (Thermo Fisher) and detected by FluorChem E system (ProteinSimple, USA). The density of bands was quantified using ImageJ software (NIH). Image has been cropped for presentation (full-size image is shown in Supplementary Figs. 12, 13).

One set of brain sections from one hemisphere of each bird was processed for both PNN and PV expression. Free-floating sections were washed 3X for 5 min in 1X TBS and then blocked for 30 min in TBS + 5% donkey serum + 0.1% Triton-X. Then the tissue was incubated overnight at 4°C in a mouse monoclonal anti-chondroitin sulfate (C8035; Sigma-Aldrich; 1:500) and a rabbit polyclonal anti-PV (ab11427; Abcam; 1:2000). Thereafter, sections were washed 3X for 5 min in TBS followed by a 2-h incubation at room temperature with secondary antibodies [donkey anti-mouse Alexa Fluor 488: 10 μl/ml (ThermoFisher); donkey anti-rabbit Alexa Fluor 594: 5 μl/ml (ThermoFisher)] in TBS + 0.1% Triton-X. The tissue was then washed 3X for 5 min in TBS and transferred to TBS before mounting. Sections were coverslipped with Prolong Gold Antifade (Life Technologies, 2491361).
Mouse brains were processed in a similar manner as finch brains, albeit with different primary antibodies (Cisneros-Franco et al., 2018 (link)). For mouse sections, PV neurons were stained using same PV antibody used in finches (1:500), while PNNs were stained using Wisteria floribunda lectin (Sigma L1516; 1:1000).

One well from each hemisphere of all birds was double-labeled for parvalbumin (PV) and chondroitin sulfate, one of the main components of the perineuronal nets, following a previously described protocol [22 (link), 37 (link), 41 (link)]. Briefly, sections were blocked in 5% Normal Goat Serum (NGS) diluted in Tris-buffered Saline (TBS) with 0.1% Triton-X-100 (TBST) for 30 minutes. They were incubated overnight at 4°C in a mixture of 2 primary antibodies diluted in TBST: a mouse monoclonal anti-chondroitin sulfate antibody (clone CS-56, 1:500 for Experiment 1 and 3 or 1:1000 for Experiment 2, C8035, Sigma Aldrich) specific for the glycosaminoglycan portion of the chondroitin sulfate proteoglycans that are the main components of the PNN and a polyclonal rabbit anti-parvalbumin antibody (1:1000; ab11427, Abcam). Sections were then incubated at room temperature in a mixture of secondary antibodies diluted in TBST. A goat anti-mouse IgG coupled with Alexa 488 (green, 1:100, Invitrogen) was used to visualize PNN staining and a goat anti-rabbit IgG coupled with Alexa 546 (red, 1:200, Invitrogen) was used to visualize PV cells. Finally, sections were mounted on slides using TBS with gelatin and coverslipped with Vectashield containing DAPI (H-1500, Vector laboratories) to confirm that PNN that were not surrounding PV-positive cells were localized around a cell nucleus.

The telencephalic tissue from the 3rd series of sections was then simultaneously immunostained for PV and chondroitin sulfate to label perineuronal nets (PNN) as described previously (Cornez et al., 2015 (link), 2017b (link), 2018b (link)) to obtain an additional measure of HVC plasticity (van 't Spijker and Kwok, 2017 (link)). Sections were rinsed 3 × 5 min in TBS and incubated in blocking solution made of 5% NGS and 0.1% Triton X-100 in TBS. Sections were then incubated overnight in a mixture of two primary antibodies including a polyclonal rabbit raised against PV (Abcam ab11427; 1:1000 in TBS-T 0.1% Triton X-100) and a monoclonal mouse anti-chondroitin sulfate antibody (1:500 in TBS-T 0.1% Triton X-100, Sigma-Aldrich C8035) for 48 h at 4°C on a rotating shaker. On the next day, sections were then washed 3 × 5 min in TBS and incubated for 2 h at room temperature on a rotating shaker in a cocktail of secondary fluorescent antibodies containing goat anti-mouse Alexa Fluor 488 (1:100, Invitrogen) and goat anti-rabbit Alexa Fluor 546 (1:200, Invitrogen). Sections were rinsed 3 × 5 min in TBS and then mounted on glass slides. Sections were dried and coverslipped with Vectashield mounting medium containing 4',6-diamidine-2'-phenylindole dihydrochloride (DAPI) to label all cell nuclei.

Immunocytochemistry was used to assess NG2 levels in Neu7 cells. Neu7 cells were seeded into eight-well chamber slides at 2000 cells per well in Neu7 cell culture medium and incubated overnight at 37 °C and 5% CO₂/90% humidity. Medium was removed, cells were washed with 1× PBS, fixed with 4% PFA and nonspecific staining was blocked as above. An antibody against NG2 (AB5320, 1:200; Merck Millipore, Darmstadt, Germany), GFAP (Z0334; 1:200; DAKO, Glostrup, Denmark) or CS-56 (C8035; 1:200; Sigma-Aldrich ((Dublin, Ireland) was added. After 24 h of primary antibody incubation, the Neu7 cells were washed with 1× PBS three times (5 min/wash). The Neu7 cells were then incubated with secondary antibodies for 2 h at room temperature, washed and cell nuclei stained as above. Cells were imaged using confocal microscopy.

For MMP-2/O4 staining, OPCs were plated onto 24-well coverslips that were precoated with PLL, 1 μg/mL laminin, and 2 μg/mL aggrecan and incubated with vehicle or 2.5 μM ISP for 2 days at 37 °C. Cultured OPC or OL cells were fixed in 4% PFA and followed by blocking in PBST solution (10% normal goat serum and 0.2% Triton–X100 in PBS). Diluted primary antibodies were incubated with samples overnight at 4 °C and followed by appropriate secondary antibody goat anti-mouse or anti-rabbit IgM or IgG conjugated with Alexa Fluor 488 or 594 (1:500, Invitrogen). The following primary antibodies were used: PDGFRα (ab65258, Abcam, 1:250), O4 (Hybridoma Core, Cleveland Clinic), MBP (SMI-99P, Covance, 1:300), MMP-2 (Ab19167, Millipore, 1:200), NG2 (AB5320, Millipore, 1:250), and CS56 (C8035, Sigma, 1:250). Cells were mounted with Vecta Shield mounting medium with DAPI (Vector Laboratories).