The middle lobe of the right lung was fixed in 4% paraformaldehyde. The tissue was completely embedded in paraffin, cut into small sections, stained with hematoxylin and eosin, and measured by optical microscopy.
For immunofluorescence staining of mouse lungs, paraffin slides were dewaxed and dewatered, followed by permeabilization with 0.5% Triton X-100 for 10 min. Endogenous peroxidase blocking solution and goat serum were used to reduce the background. Then, the cells were stained with donkey polyclonal anti-human IL-33 (1:50, R&D, Minneapolis, MN, USA), rabbit polyclonal anti-mouse EpCAM (1:50, Abcam, Shanghai, China), anti-mouse SFTPC (1:50, Abcam, Shanghai, China), and anti-mouse CD31 (1:50, Abcam, Shanghai, China). Images were acquired with an Olympus Fluoview confocal microscope.
IL-33-positive nuclei were quantified automatically as the fraction of DAPI+ structures exhibiting IL-33 staining using ImageJ. The following settings were used: type-8 bit, invert, subtract background, threshold (80, 255), watershed, and analyze particles (size = 50, Infinity circularity = 0.1–1.00).
For immunofluorescence staining of mouse lungs, paraffin slides were dewaxed and dewatered, followed by permeabilization with 0.5% Triton X-100 for 10 min. Endogenous peroxidase blocking solution and goat serum were used to reduce the background. Then, the cells were stained with donkey polyclonal anti-human IL-33 (1:50, R&D, Minneapolis, MN, USA), rabbit polyclonal anti-mouse EpCAM (1:50, Abcam, Shanghai, China), anti-mouse SFTPC (1:50, Abcam, Shanghai, China), and anti-mouse CD31 (1:50, Abcam, Shanghai, China). Images were acquired with an Olympus Fluoview confocal microscope.
IL-33-positive nuclei were quantified automatically as the fraction of DAPI+ structures exhibiting IL-33 staining using ImageJ. The following settings were used: type-8 bit, invert, subtract background, threshold (80, 255), watershed, and analyze particles (size = 50, Infinity circularity = 0.1–1.00).