Anti sftpc

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.