Anti sox2

Lungs from AdenoCre-induced K-Ras^G12D/+;Ahr^+/+ and K-Ras^G12D/+;Ahr^−/− mice were fixed in buffered formalin, deparaffinated and rehydrated in PBS. Antigen unmasking was performed by incubation in citrate buffer at pH 6. Sections were washed in PBS containing 0.05% Triton X-100 (PBS-T) and non-specific epitopes blocked in PBS-T containing 0.2% gelatin and 3% BSA (PBS-T-G-B) for 1 h at room temperature. Sections were then incubated overnight at 4 °C with the following primary antibodies diluted in PBS-T-G-B: anti-SFTPC (Millipore 1:200, Temecula, CA, USA), anti-SCGB1A1/CCL10 (Novus Biologicals, 1:100, Abingdon, UK), anti-SOX2 (Novus Biologicals 1:150), anti-ALDH1A1 (Abcam 1:200), anti-EPCAM (Santa Cruz Biotechnology 1:200), anti-LGR5 (Origene 1:200) and anti-PORCN (Millipore 1:150). Following washing in PBS-T, sections were incubated for 1 h at room temperature with Alexa-488, Alexa-550 or Alexa-633-labeled secondary antibodies diluted in PBS-T-G-B. After additional washing, sections were dehydrated and mounted in PBS:glicerol (1:1). Visualization was done in an Olympus FV1000 confocal microscope. Fluorescence analysis was done using the FV10 software (Olympus, Shinjuku, Japan) and Image J software. DAPI was used to stain cell nuclei. At least three replicates were performed of each condition analyzed. For each replicate, four to six fields were measured at the microscope.

The cells were rinsed briefly with phosphate-buffered saline (PBS) and fixed for 20 min in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) at room temperature. The cells were permeabilized for 10 min with 0.1% Triton X-100 in PBS, and blocked for 45–60 min with 4% bovine serum albumin in PBS at room temperature. Cells were incubated overnight at 4 °C with one of the following antibodies: anti-Oct4 (1:500; Abcam, Cambridge, MA, USA), anti-Sox2 (1:500; NB110-37235, Novus Biologicals, Littleton, CO, USA), anti-Nanog (1:500; Abcam, Cambridge, MA, USA), anti-c-Myc (1:250; bs-4963R, Bioss, Woburn, MA, USA), anti-Klf4 (1:250, bs-1064R, Bioss, Woburn, MA, USA). This was followed by incubation with the following secondary antibody: Alexa Fluor 488-labeled anti-rabbit IgG (1:500; Invitrogen, Carlsbad, CA, USA). Nuclei were counterstained using DAPI (1 mg/mL PBS; Invitrogen, Carlsbad, CA, USA).