Ab87117

Adult mouse astrocytes in culture were lysed using RIPA buffer which included halt protease and phosphatase inhibitor cocktail (catalogue no. 78443, Thermo Fisher Scientific), followed by centrifugation at 14,000× g at 4 °C for 15 min. Then, 30 μg of protein was mixed with Laemmli sample buffer (NP0007, Invitrogen) at 90 °C for 5 min. Proteins were separated on 4–20% gradient Tris-Glycine polyacrylamide gels (Thermo Fisher Scientific), followed by transfer onto nitrocellulose membranes by the iBlot2 Dry Blotting System (Thermo Fisher Scientific). Membranes were incubated with blocking buffer (927-6001, LI-COR), followed by incubation with primary antibodies against excitatory amino acid transporter 2 (EAAT-2) (ab41621, Abcam), vimentin (ab92547, Abcam), and aldehyde dehydrogenase 1 family member L1 (ALDH1L1) (ab87117, Abcam) at 1:1000 dilution at 4 °C overnight at room temperature. After washing with 0.5% Tween-20 TBS, the membranes were incubated with IRDye^® 800CW donkey anti-rabbit IgG (926-32213, LI-COR) secondary antibody at 1:2000 dilution for 1 h. Images were captured by LI-COR ODYSSEY CLx.

Immunoblotting was performed as previously described.^{55 (link)} Briefly, tissues were lysed in lysis buffer and protein concentration was determined by Bradford. Proteins were separated on polyacrylamide gel and transferred to PVDF membrane. The membrane was blocked in 5% milk and subsequently probed with primary antibodies overnight at 4 °C, then incubated with HRP-conjugated secondary antibodies for protein detection. The antibodies used were anti-GFAP (Z0334, Dako, Santa Clara, CA,USA), anti-ALDH1L1 (Ab87117, Abcam, Cambridge, UK), anti-MBP (78896, Cell Signaling Technology, Cambridge, MA, USA), anti-Olig2 (AB9610, Millipore, Billerica, MA, USA), anti-Prrc2a (Sc-78859, Santa Cruz, Dallas, TX, USA), anti-FLAG (F1804, Sigma), anti-Myc (M047-3, MBL, Woburn, MA,USA), anti-Ythdf1 (17479-1-AP, Proteintech Group, Campbell Park, Chicago, IL,USA), anti-Ythdf2 (24744-1-AP, Proteintech Group), anti-PARP1 (ab191217, Abcam), anti-β-actin (60008-1-Ig, Proteintech Group), anti-GAPDH (CW0266A, CWBiotech, Beijing, China), and anti-β-tubulin (CW0098A, CWBiotech). Polyclonal rabbit anti-FTO antibody was affinity-purified from rabbits immunized with 6 × His tagged full-length human FTO protein as previously reported.^{5 (link)} Polyclonal rabbit anti-ALKBH5 antibody was generated against synthesized peptide by CWBio (Beijing) as previously reported.^{4 (link)}

Totally, 80 (embryos and pups) or 60 (adults) µm mouse brains sections were cut with a vibrating-blade microtome (VT1000S, Leica) following embedding in 3% agarose dissolved in PBS 1X. Immunohistology or EdU revelation (Click-iT^™ Plus EdU Cell Proliferation Kit for Imaging, Alexa Fluor^™ 647 dye, Sigma) were performed on free floating sections after 1 h incubation in a 0.2% gelatin, 0.5% Triton X-100 (Sigma) PBS 1X blocking solution. Sections were then incubated O/N (embryos) to 72 h (adults) at room temperature with 1:400 rabbit (Merck Millipore AB9610) or 1:200 goat (R&D systems Bio-Techne AF2418) anti-Olig2, 1:500 rabbit anti-Aldh1l1 (Abcam ab87117), 1:500 rabbit anti-S100β (Abcam ab52642), and 1:500 rabbit anti-Ki67 (Abcam ab15580) primary antibodies diluted in blocking solution, then rinsed 3 × 5 min with PBS 1X and incubated 3 h at room temperature with 1:500 goat anti-rabbit Alexa Fluor 647 (Life A21245), 1:500 donkey anti-goat Alexa Fluor 594 (Life A11058) or 1:500 donkey anti-rabbit Alexa Fluor 647 (Jackson 711-605-152) species-specific secondary antibodies. All samples were rinsed 3 × 5 min with PBS 1X and mounted in Vectashield mounting medium (Vector Labs).

Immunofluorescence stainings were carried out as described in [32 (link)]. Primary antibodies used were anti-β-III-tubulin (mouse; 1:2000, G7121, Promega, Madison, WI, USA), anti-MAP2 (chicken; 1:5000, ab5392, Abcam), anti-VGLUT1 (rabbit; 1:500, ab180188, Abcam, Cambridge, UK), anti-NeuN (rabbit; 1:500, ab177487, Abcam, Cambridge, UK) anti-ALDH1L1 (rabbit; 1:500, ab87117, Abcam), anti-EAAT1 (rabbit; 1:200, ab416-1001, Abcam, Cambridge, UK) anti-GFAP (mouse; 1:400, C53893, Sigma-Aldrich, St. Louis, MO, USA), and anti-connexin 43 (mouse; 1:500, 14-4759-82, Invitrogen, Carlsbad, CA, USA). Secondary antibodies used were anti-mouse Cy3 (1:1000, Thermo Fisher Scientific), anti-rabbit 488, and anti-chicken Cy5 (1:1000, Abcam). Nuclei were stained with DAPI (1:1000, Sigma-Aldrich), and coverslips were mounted using Dako Fluorescing Mounting Medium.

PF-PBS post-fixed retina, lamina and ON frozen sections were analyzed for fluorescent dendrimer signal (D-Cy5), as well as for RGCs (goat anti human Brn3a; Santa Cruz Sc-31984; 1:500 concentration), total inflammatory cells (mouse anti-human IBA1; Millipore MABN92; 1:500 concentration), and astrocytes (glial acidic fibrillary protein [rabbit anti-pig GFAP; Sigma-Aldrich clone GA5; 1:1000 concentration] and aldolase dehydrogenase 1L1 [rabbit anti-mouse Aldh1L1; Abcam ab87117]; 1:1000 concentration). We performed antigen recovery using citrate buffer. Primary antibodies made in different species (mouse antihuman IBA1 and rabbit antimouse Aldh1L1) enabled us to colocalize both Aldh1L1 and IBA1 without difficulty, using fluorescently labeled donkey anti-rabbit (Cy2) and donkey anti-mouse (Cy3) secondary antibodies. Histological analysis was performed using a 4-channel confocal microscope (Olympus E900).

Dorsal and ventral spinal cord (Figure 1E) samples were microdissected from acute slices prepared in the same manner as done for electrophysiology (see below). Sample lysis was performed in RIPA buffer (Thermofisher) in the presence of protease and phosphatase inhibitors (Cell signaling). Samples concentration was determined with the Bradford method and protein migration and gel transfer was performed as described previously (Kenney and Rowitch, 2000 (link)). After blocking in Odyssey Blocking Buffer (PBS) (Li-Cor) for 1 hr, RT, primary antibodies were incubated O/N at 4°C onto the western blot membrane. The following antibodies were used: rabbit Kir4.1 (extracellular, APC-165, Alomone, 1:2000), rabbit Kir4.1 (APC035, Alomone, 1:2000), mouse GFAP (Sigma, 1:500), rabbit ALDH1L1 (ab87117,Abcam, 1:500), mouse β-actin (Sigma, A5441, 1:5000 or AC-74, 1:20000). IRDye Goat anti-mouse and anti-rabbit (680 and 800) fluorescent secondary antibodies (Li-Cor) were used for protein detection on the Odyssey Cxl imaging system.