Dual Immunofluorescence Staining of Lung Cells

Fixation of cells was conducted as described above. Permeabilization was conducted with 0.25% Triton X-100/PBS for 7 min at RT. Double staining of target antigens was achieved by incubation with first primary antibody for 45 min. Followed by subsequent washing with PBS and incubation with the second primary antibody at the same conditions. Visualization of primary antibodies was conducted with Alexa488 or TRITC labeled secondary antibodies (Molecular Probes, Eugene, Oregon, USA) diluted at 1/200 with 1 μg/ml DAPI in antibody diluent (Zytomed Systems, Berlin, Germany) for 45 min. at room temperature. Unbound antibodies and DAPI were removed by washing with PBS before mounting of cover slips on microscopic slides (Super Frost Plus, Menzel Gläser, Germany) with DABCO. In general, each reagent step was followed by thorough washing with 200 μl of PBS. To detect cells of hAECII lineage, the surfactant protein transcription factor TTF-1 [19 (link)] was targeted with mouse anti-TTF-1 (clone SPT24, Zytomed Systems, Berlin, Germany) at 1/400 dilution. Activity of collagen production and secretion processes were assessed by targeting the collagen chaperon HSP47 [20 (link)] with rabbit anti-HSP47 (clone EPR4217, Abcam, Oxford, UK) at 1/100 dilution.