Goat anti sox9

Mice were perfused with phosphate-buffered saline (PBS) and then 4% paraformaldehyde (PFA) in PBS. The brain was dissected and postfixed overnight in 4% PFA-PBS. Mouse brains were soaked in 30% sucrose-PBS for cryoprotection. Brains were sectioned into 40 μm thick slices using a sliding microtome (Leica). Brain sections were blocked in 0.3% Triton X-100 and 10% normal horse serum before overnight incubation in primary antibody at 4 °C. The following primary antibodies were used: guinea pig anti-Iba1 (1:500; Synaptic System, Göttingen, Germany, #234308), goat anti-SOX9 (1:2000; R&D Systems, Minneapolis, MN, USA, #AF3075), mouse anti-GFAP (1:500; Cell Signaling Technology, Danvers, MA, USA, #3670S), mouse anti-Olig2 (1:250; Millipore, Burlington, MA, USA, #MABN50), rabbit anti-ASPA (1:1500; GeneTex, Irvine, CA, USA, #GTX113389), chicken anti-GFP (1:2000; abcam, Cambridge, UK, #13970), and rabbit anti-DsRed (1:1000; TaKaRa, San Jose, CA, USA, #632496). The brain sections were washed three times with PBS before incubation in secondary antibody for 1 h at room temperature. The brain sections were then counterstained with DAPI, mounted onto microscope slides, and covered with mounting media.

Differentiated cells at specific stages were fixed with 70% ethanol and blocked overnight with 6% BSA in PBST. Cells were stained with the primary antibodies goat anti-PDX1 (1:100; Abcam), goat anti-SOX9 (1:50; R&D Systems), mouse anti-NKX6.1 (1:100; DSHB), goat anti-NKX6.1 (1:50; R&D Systems), and mouse anti-Ki67-647 conjugate (1:50; BD Biosciences) for 3–4 h at room temperature. The cells were incubated with Alexa-fluor secondary antibodies (1:200; Molecular Probes, ThermoFisher Scientific) for 40 min at room temperature. The results were analyzed using the BD Accuri C6 flow analyzer and the results were processed using FlowJo.

Brain sections were treated overnight with primary antibody in 5% normal donkey serum/PBS with 0.1% Tween-20 at 4 °C and followed by the appropriate fluorescently conjugated secondary antibody. For samples requiring further unmasking of the epitope, which included anti-Aldh1L1, antigen retrieval was performed by boiling in 6 M sodium citrate buffer at pH 6.0 for 20 min. Slides were then cooled to room temperature and washed 3× with PBS and 0.1% Tween-20, and the standard immunostaining protocol was followed. Well characterized primary antibodies were as follows: rabbit anti-Iba1 (1:500, WAKO), goat anti-Iba1 (Abcam; 1:300), rat anti-SF1 (1:800, kindly provided by Dr. Taro Tachibana, Osaka City University JAPAN), mouse anti-Olig2 (1:300, Millipore), rat anti-LAMP1 (1:800, Millipore), rat anti-CD68 (1:500, BIORAD), goat anti-Sox9 (1:60, R&D systems), rabbit anti-NKX2.1 (1:400, Santa Cruz), sheep anti-Csf1R (1:300, R&D Systems), goat anti-PdgfR alpha (1:150, R&D Systems), rabbit anti-GFAP (1:500; DAKO), rabbit anti-SB100 (1:400; DAKO), and rabbit anti-Aldh1l1 (1:500, Abcam). All appropriate secondary antibodies were Donkey anti-IgG Alexa Fluor conjugated (1:400, ThermoFisher Scientific). All samples were counterstained with Hoechst nuclear stain (1:1000; ThermoFisher Scientific H3570).