Ab54583

Lungs from wild type mouse embryos from E11 till E18 were dissected and fixed in 4% PFA overnight at 4°C before processing for paraffin embedding. Sections of 5 μm were dewaxed and rehydrated, followed by antigen retrieval with microwave treatment in TE. Samples were blocked and incubated with antibodies against CHD4 (ab72418) and SOX2 (GTI5098), FOXP2 (ab16046) and SOX2, and CUX1 (Abcam, Ab54583) and SOX2 diluted in blocking buffer at 4°C overnight. After washing in PBS with 0.05% Triton X-100, slides were incubated in blocking buffer with fluorophore conjugated secondary antibody (Alexa Fluor-488, Alexa Fluor-594; Jackson Immuno Research) for 1 h at room temperature. Slides were mounted in Vectashield Mounting Medium with Dapi (Vector laboratories, Burlingame, CA, United States). Digital images were captured using a ZEISS imager Z1 AX10 microscope.

Antibodies for CTIP2 (ab18465, 1:500), CUX1 (ab54583, 1:500), PE-conjugated anti EGFR (ab231, 1:50), LGALS1 (ab25138, 1:1,000), Lin28 (ab46020, 1:1,000), PHH3 (ab5176, 1:250), PLZF (ab104854, 1:100), POU3F2 (ab94977, 1:1,000), SATB2 (ab51502, 1:50), SOX1 (1:1,000), SOX2 (ab79351, 1:500), TBR1 (ab31940, 1:200), TBR2 (ab23345, 1:200) were from Abcam. Antibodies for BrdU (347580, 50 μl per test), KI67 (556003, 1:1,000), phycoerythrin-conjugated (PE) SSEA-3 (560237, 20 μl per test), PE-conjugated F11R (552556; 20 μl per test), Alexa Fluor 647-conjugated TRA-1-60-647 (560850, 5 μl per test), Alexa Fluor 647-conjugated TUJ1 (560340, 1:500) were from BD Biosciences. Antibodies for DCX (AB2253, 1:5,000), O4 (MAB345, 1:25), RELN (MAB5364, 1:200), Tyrosine Hydroxylase (TH, AB152, 1:500) were purchased from Millipore. Antibodies for FABP7 (51010-1-AP, 1:100), S100B (15146-1-AP, 1:100) were from ProteinTech. Antibodies for AP2α (3B5 concentrated, 1:100) and PAX6 (supernatant, 1:16) were from DSHB. Antibody for NESTIN (MO15012, 1:500) was from Neuromics. Antibody for GFAP (Z0334, 1:2,000) was from DAKO. Antibody for β-3-Tubulin (PRB-435P, 1:1,000) was from Covance. Antibody for GLAST (ACSA-1; 130-095-822, 1:10) was from Miltenyi Biotec. Antibody for OTX2 (AF1979; 1:40) was from R&D. Antibody for FOXA2 (SC-6554; 1:100) was from Santa Cruz Biotechnology.

Lungs from wild type mouse embryos from E11 till E18 were dissected and fixed in 4% PFA overnight at 4°C before processing for paraffin embedding. Sections of 5 μm were dewaxed and rehydrated, followed by antigen retrieval with microwave treatment in 10 mM Tris-HCl, 1 mM EDTA pH9.0 (TE). After quenching endogenous peroxidase with 1.5% H₂O₂, samples were blocked with 1% BSA, 0.05% Tween20 in PBS for 10 min at room temperature and then incubated with antibody against CHD4 (Abcam; ab72418), FOXP2 (Abcam; ab16046), FOXP4 (Santa Cruz; sc-292474), or CUX1 (Abcam, Ab54583) diluted in blocking buffer at 4°C overnight. After washing in PBS with 0.05% Tween20, slides were incubated in blocking buffer with biotin conjugated secondary antibody for 30 min at room temperature. Then samples were incubated with VECTASTAIN^® ABC Reagent (Vector) for 30 min, followed by incubation with diaminobenzidine (Fluka). Slides were counterstained with hematoxylin and mounted with pertex. Sections were analyzed using Olympus BX41 microscope.