Rabbit anti nkx2

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).

Pregnant females were euthanized by lethal intraperitoneal (i.p.) injection of pentobarbital (50 mg kg⁻¹), embryos were collected by caesarian cut and brains were dissected and fixed ON in cold 4% paraformaldehyde (PFA) dissolved in 0.1 M phosphate buffer, pH 7.4. For postnatal brains, animals were deeply anaesthetized by i.p. injection of pentobarbital, transcardially perfused with 0.9% saline followed by cold 4% PFA and postfixed (ON) in cold 4% PFA. Brains were cut on a Vibratome (Leica, VT1000S) for IHC and for free-floating in situ hybridization. Sections were kept at 4 °C in 0.1 M phosphate buffer saline and were stained by IHC as described31 (link) with the following primary antibodies rabbit anti-GFP (1:500; Millipore), goat anti-GFP (1:1,000; Chemicon), mouse anti-human ovalbumin upstream promoter transcription factor 2 (COUP-TFII; 1:500; Perseus proteomics), goat anti-SP8 (1:50; Santa-Cruz), rabbit anti-NKX2.1 (1:100; Santa-Cruz), mouse anti-PV (1:1,000; Swant), rat anti- SST (1:500; Millipore), mouse anti-Reelin (1:500; Abcam), rabbit anti-VIP (1:500; Abcam), rabbit anti-NPY (1:1,000; Immunostar), rabbit anti-Calretinin (1:1,000; Swant). Secondary goat or donkey Alexa-488, -568 and -647 antibodies (Molecular Probes, Invitrogen) raised against the appropriate species were used at a dilution of 1:500–1,000 and sections were counterstained with Hoechst 33258 (1:10,000).

The following antibodies were used: rat anti-CDH1 (1:200, Santa Cruz, sc-59778); mouse anti-CDH1 (1:100, BD Biosciences, 560062); rabbit anti cleaved caspase-3 (1:600, Cell Signaling Technologies, #9661); goat anti-SCGB1A1 (1:200, Santa Cruz, T-18); rabbit anti-NKX2.1 (1:400, Santa Cruz, H-190); mouse anti-acetyl-α tubulin (1:2000, Sigma, MABT868); mouse anti-FOXJ1 (1:400, Thermo Fisher Scientific, 14-9965-82); rabbit anti-MUC5AC (1:400, Santa Cruz, H-160); rabbit anti-MUC5B (1:400, Novus Biologicals, NBP1-92151); mouse anti-BrdU (1:400, Thermo Fisher Scientific, B35141); hamster anti-PDPN (1:20, DSHB, 8.1.1); rat anti-SCGB3A1 (1:200, R&D Systems, MAB2954); rat anti-SCGB3A2 (1:200, R&D Systems, MAB3465); rabbit anti-Ki67 (1:400, Thermo Fisher Scientific, PA5-19462); goat anti-NGFR (1:200, Santa Cruz, C-20); mouse anti-α-SMA-Cy3 (1:1000, Sigma-Aldrich, C6198); rabbit anti-SOX9 (1:400, Millipore, AB5535MA); goat anti-SOX9 (1:500, R&D Systems, AF3075); rat anti-Cd140a (1:100, Biolegend, 135901); rabbit anti-RFX3 (1:500, Sigma, HPA035689); mouse anti-TRP63 (1:500, Abcam, ab735); rabbit anti-TRP63 (1:100, Cell Signaling Technology, 13109S) and chicken anti-KRT5 (1:400, Biosite, 905901).