Immunofluorescence Staining of COPD Lung Tissue

Immunofluorescence staining was performed on paraffin-embedded sections of human lung tissue of a COPD GOLD II patient. The sections were subjected to antigen retrieval using EDTA buffer (Dako). After washing steps with PBS, the sections were incubated with Boehringer blocking agent (Roche) with 0.3% Triton X-100 (Sigma-Aldrich). Primary antibodies were added for 24 hours: rabbit anti-BPIFA1, rabbit anti-BPIFB1,9 (link) mouse anti-MUC5AC (Abcam), mouse anti-P63 (Abcam), isotype rabbit IgG (Novus Biologicals, Littleton, CO, USA), isotype mouse IgG2a (BD Biosciences, San Diego, CA, USA), and isotype mouse IgG1 (BD Biosciences). Wash steps were performed, and the sections were incubated with secondary antibodies: donkey anti-rabbit Alexa Fluor 555 (Thermo Fisher Scientific, Waltham, MA, USA), donkey anti-mouse Alexa Fluor 488 (Thermo Fisher Scientific), and DAPI (Thermo Fisher Scientific). Finally, the sections were mounted with fluorescence mounting medium (Dako). Images were recorded with the Axiovision software from Zeiss. BPIFA1 and BPIFB1 were visualized as green, whereas MUC5AC and P63 were visualized as red.