Goat anti scgb1a1

Lung tissues were routinely perfused, inflated, and fixed in formalin for 2 h at room temperature. Culture colonies were fixed with 4% paraformaldehyde for 1 h at 4 °C, immobilized in O.C.T. compound (Tissue-Tek; Sakura, Torrance, CA, USA), and frozen with liquid nitrogen. Paraffin sections (5 μm) and cyrosections (6 μm) were used for staining. Antigen retrieval was performed in citric acid (10 mM; pH 6), followed by blocking with 5% BSA in 0.2% Triton-X/PBS for 30 min at room temperature. Primary antibodies were incubated overnight at 4 °C at the indicated dilutions: mouse anti-E-cadherin (1:50; Invitrogen), goat anti-Scgb1a1 (1:200; Santa Cruz Biotechnology, Dallas, TX, USA), mouse anti-CYP2F2 (1:100; Santa Cruz Biotechnology), and rabbit anti-Ki67 (1:200; eBioscience). Fluorochrome-conjugated secondary antibodies (1:200; Invitrogen) were incubated at room temperature for 90 min. After antibody staining, sections were mounted with Fluoromount G containing 4′-6′-diamidino-2-phenylindole (DAPI). Stained sections were imaged using a Leica TCS SP5 confocal microscope (Leica, Wetzlar, Germany). The number of E-cadherin⁺ and Ki67⁺ cells was counted in three or more random 20 × views of each colony section, and the percentage of E-cadherin⁺Ki67⁺ cells in the total E-Cadherin⁺ cell population of each colony section was calculated and averaged.