Vectastain elite abc and dab substrate kits

Tissues were fixed in 4% paraformaldehyde or 10% formalin, paraffin embedded and analyzed by hematoxylin and eosin staining. Immunohistochemistry was performed using Vectastain Elite ABC and DAB Substrate kits (Vector Laboratories) or the automated BenchMark XT IHC/HIS Staining Module. Methanol/hydrogen peroxide pretreatment and serum blocking were performed, and some antibodies were applied after microwave 10 mmol/L citrate, EDTA, or protease antigen retrieval. Antibodies and dilutions are detailed in Supplemental methods. Slides were counterstained with nuclear fast red or hematoxylin.

Tissues were fixed in 10% formalin, paraffin embedded and analyzed as hematoxylin and eosin (H&E) stained sections. Methanol/hydrogen peroxide pretreatment, microwave 10mM citrate antigen retrieval, and serum blocking was performed. Antibodies were incubated at 4°C overnight: CALCA (Sigma-Aldrich, C8198, 1:8000), SCGB1A1 (Santa Cruz Biotechnology Inc., sc-9773, 1:2000), SFTPC (pro-SPC) (Seven Hill, RB458, 1:3000), NKX2-1 (Seven Hill, 1231, 1:1000), and SOX2 (Seven Hill, R1236, 1:200). For immunohistochemistry, slides were stained with Vectastain Elite ABC and DAB Substrate kits (Vector Laboratories) and counterstained with nuclear fast red. For immunofluorescence, slides were incubated with β-galactosidase (Abcam, ab9396, 1:1000) and CALCA (Sigma-Aldrich, C8198, 1:8000) followed by goat anti-chicken Alexa Fluor 484 (Invitrogen, A11039, 1:200) and goat anti-rabbit Alexa Fluor 594 (Invitrogen, R37117, 1:200) secondary antibodies for 1 hour at room temperature in the dark. Slides were coverslipped with Vectashield mounting medium with DAPI (Vector Laboratories) and analyzed using the Zeiss Apotome system (Zeiss, Jena, Germany).