Licor odyssey tbs blocking buffer

Total RNA was obtained using TRIzol (Invitrogen), treated with DNAse I (DNA-free, Ambion, Grand Island, New York, USA), and cDNA produced using SuperScript III Reverse Transcriptase (Invitrogen). For cDNA generated from FACS isolated MaSC-enriched, luminal progenitor and mature luminal populations, a pre-amplification step was performed following the TaqMan® (Applied Biosystems, Grand Island, New York, USA) PreAmp Master Mix protocol as per manufacturer's instructions. qRT-PCR was performed using TaqMan® Gene Expression Assays and Roche Universal Probe Library primers (listed in Supplementary Methods). Sample quality was verified by comparing C_t values for Gapdh. For immunoblotting, fibroblasts were lysed on ice [50mM Tris-HCl, pH7.4; 100mM NaCl; 1mM EDTA; 1mM EGTA; 1mM NaF; 0.1% SDS; 0.5% Sodium Deoxycholate; 1% Triton-X-100; 10% Glycerol; Protease and Phosphatase Inhibitor Cocktails (Sigma)], and protein levels quantified (Bradford Assay, Bio-Rad, Hercules, California, USA). Protein lysate was resolved using SDS-PAGE, and transferred to PVDF membrane (EMD Millipore, Billerica, Massachusetts, USA). The LiCOR Odyssey TBS Blocking Buffer (Lincoln, Nebraska, USA) was used to block and as a diluent for both primary (Jagged-1-#2620, Cell Signaling; β-actin-#A1978, Sigma) and secondary antibodies (LiCOR). Signal was detected using the LiCOR Odyssey®.