Prolong gold antifade solution containing dapi

The following antibodies and reagents were used for paraffin embedded mouse lung tissues, as well as in vitro formalin fixed cell culture: F-Actin-488 conjugated (1:250 IF, A12379; Life Technologies Corporation); Collagen-1 (1:100 IF, 1:750 WB, NB600–408; Novus Biotechnologies); α-SMA (1:300 IHC, AB5694; Abcam); pSmad3 (1:400 IHC, 118825; Abcam); Hyaluronic Acid Binding Protein (1:100 IHC, 385911; Millipore); EZLink Maleimide-PEG₂-Biotin (21901BID; Thermo Scientific); Anti-Human IL-10 (1:200 IHC, ab9969;Abcam); STAT3 (1:500; Cell Signalling Technology, cat. #12640); Prolong Gold Antifade Solution containing DAPI (Life Technologies Corporation).

For healthy and IPF human fibroblast cell culture staining, the following steps were performed: once the experiment had concluded, cells were washed with PBS three times followed by fixation by 4% paraformaldehyde for 10–15 minutes. Following fixation, cells were then permeabilized with PBS containing 0.1% Triton X-100 (PBST) and blocked with normal goat serum for 1 hour at room temperature, followed by primary antibody incubation overnight at 4°C. Th e next day, cells were washed again with PBST and incubated with 488, 594, or 647 fluorophore containing secondary antibodies for 1 hour at room temperature (Life Technologies Corporation). Slides were mounted with Prolong Gold Antifade Solution containing DAPI (Life Technologies Corporation).