Ab5694

For paraffin embedded mouse lungs, mouse right ventricles were perfused with 1 mL PBS and the lungs were inflated with 4% PFA, and then fixed in 4% PFA overnight at 4°C. After fixation, the lungs were washed by cold PBS 4 times in 2 hours at 4°C and dehydrated in a series of increasing ethanol concentration washes (30%, 50%, 70%, 95%, and 100%). The dehydrated lungs were incubated with Xylene for 1 hour at room temperature (RT) and with paraffin at 65°C for 90 minutes 2 times, and then embedded in paraffin and sectioned. Human and mouse PCLS samples were fixed in 4% PFA for 30 minutes. After PBS washes, slices were embedded in OCT after 30% sucrose incubations. Cryosections measuring 6–8 μm thick were used for IHC. The following antibodies were used: GFP (1:400, Abcam, ab6673), α smooth muscle actin (1:200, Abcam, ab5694), Transgelin (1:200, Abcam, ab14106), and Collagen I (1:200, Abcam, ab21286). Human lung fragments were fixed and processed as the mouse lungs. Antibodies used for human lung slide staining were ACTA2 (1:200, Abcam, ab5694), p16^INK4a (1:200, Santa Cruz, sc-56330), and CTHRC1 (1:200, Abcam, ab85739). Collagen staining was performed using Trichrome stain kit according to the manufacturer’s protocol (Abcam, ab150686). Images were captured using Zeiss Imager M1 or Leica Stellaris 5.

Cells were fixed with 4% paraformaldehyde for 20 min, washed with PBS, and then incubated with Triton X-100 for 10 min (0.5% v:v). Blocking was done by incubating with 5% (w:v) bovine serum albumin (BSA) for 30 min, washed with PBS, and then incubated with primary antibody overnight at 4 °C. Anti-α-SMA (Abcam, ab5694, 1:100, Cambridge, UK), anti-CD31 (Santa Cruz, SC1506, 1:100, Dallas, TX, USA) and vE-cadherin (Arigo-ARG11036, 1:100, Hsinchu, Taiwan) primary antibodies were used. After binding to the primary antibodies, cells were further incubated for 1h with Alexa Fluor-conjugated anti-rabbit and anti-mouse (Jackson Immunoresearch, 1:500, West Grove, PA, USA). Finally, cells were stained and mounted using the Prolong^®Diamond Antifade Mounting Medium containing DAPI (Life Technology, Carlsbad, CA, USA). Cells were analyzed microscopically with an Olympus DP71 device by magnification of 100 times and 200 times, or the Re-scan confocal microscope (Confocal.nl, Amsterdam, The Netherlands) at 600 times magnification.

Four percent paraformaldehyde (PFA, Merck) was used to fix cells for 20 min at room temperature. Fixated cells were put in permeabilization solution containing 0.2% Triton-X100 (Merck) for 15 min. Blocking solution consisted of 10% normal goat serum (NGS, Vector Laboratories) was added for 1 h. Cells were incubated in primary antibody solution containing OCT4 (1:400, Abcam, ab19857), CD31 (1:200, BioLegend, 303,110), αSMA (1:200, Abcam, ab5694), or OCN (1:400, Santa Cruz, sc-74495), or TIE-2 (1:400, Santa Cruz, sc-293414) antibody in 0.1% Triton-X100, 5% NGS overnight. Cells were washed for secondary antibody against mouse (1:400, Thermo Fisher, A21151) or rabbit (1:400, Thermo Fisher, A11008) for 1 h. Nuclei were stained with DAPI (1:200, Merck, D9541) for 15 min. For immunostaining protocol on histological samples, antigen was retrieved with proteinase K (1:100, Merck) solution before permeabilization. Visual imaging was carried out with confocal laser scanning microscopy, Eclipse Ti2 inverted-microscope (Nikon), or super-resolution microscope (SRM, Carl Zeiss). Image analysis such as merging RFP, GFP, and DAPI channels or measuring mean fluorescence intensity was carried out with ImageJ (NIH).