Slide glasses

At 30 min post-irradiation, cells seeded on coverslips were fixed with 3% paraformaldehyde (Sigma-Aldrich)–2% sucrose (Sigma-Aldrich) for 10 min at room temperature. The fixed cells were permeabilized for 3 min at room temperature with 0.2% Triton (Sigma-Aldrich) and washed with phosphate buffered saline ([PBS] Sigma-Aldrich). The cells were incubated for 30 min at 37°C with a primary antibody specific for γH2AX (05-636, Millipore, Burlington, MA, USA) prepared in 2% bovine serum albumin ([BSA] Sigma-Aldrich)–PBS. The cells were then incubated for 30 min at 37°C with a secondary antibody conjugated to Alexa-Fluor-488 (Thermo Fisher Scientific, Waltham, MA, USA) prepared at 1:500 in 2% BSA–PBS containing 0.1 mg/mL 4′,6-Diamidino-2-Phenylindole, dihydrochloride (DAPI; Roche, Mannheim, Germany). After washing with PBS, coverslips were mounted on slide glasses (Matsunami, Osaka, Japan) using Vectashield (Vector Laboratories, Burlingame, CA, USA).

Anesthetic MS-222, water-soluble progesterone, and ATP Bioluminescence Assay Kit CLS II were purchased from Sigma (St. Louis, MO). Collagenase (280 U/mg) was obtained from Wako (Osaka, Japan) and human chorionic gonadotropin was from Teikoku Zoki (Tokyo Japan). Hydrogen peroxide, Sudan Black B (SBB), and protein assay CBB solution were from Nacalai Tesque (Kyoto, Japan). Hydrogen peroxide colorimetric/fluorometric assay kit was from BioVision (Milpitas, CA). Other chemicals were obtained from Wako and Nacalai Tesque. Slide glasses and cover slips for microscopy were purchased from Matsunami Glass (Osaka, Japan).

Monkey kidney-derived fibroblast-like cells (Cos7 cells) purchased from the Cell Engineering Division in RIKEN BioResource Centre (RBC0539; Ibaraki, Japan) were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% foetal bovine serum at 37 °C with 5% CO₂. For HS-AFM observation, slide glasses (Matsunami Glass Ind., Ltd., Osaka, Japan) or 0.15 CG pierced slide glass (ToA Optical Technologies, Inc., Tokyo, Japan) were coated with 0.01% poly-L-lysine solution (Sigma-Aldrich) and incubated at 37 °C for 1 h. Cos7 cells were seeded on the glass, incubated overnight, and subjected to HS-AFM observation the next day. CK666 (Abcam, Cambridge, United Kingdom) was added to the culture medium to a final concentration of 100 µM 30 min before the observation. Polyethyleneimine (M.W. 40,000, Polyscience, Niles, IL, USA) was used to introduce the expression vectors into Cos7 cells.

Detection of IL-33 and TSLP by immunohistochemistry was performed as described elsewhere with minor modifications^{29 (link)}. Twenty-four hours after the last chitin inhalation, the trachea was cannulated with a 22-G blunt needle attached to a syringe, and 4% paraformaldehyde in 0.05 M phosphate buffer (pH 7.4) was infused. The lungs were immediately harvested and immersed in a fixative solution consisting of 4% paraformaldehyde in 0.05 M phosphate buffer (pH 7.4) at 4 °C for 20–24 h. The tissues were then immersed in 30% sucrose in 0.05 M phosphate buffer (pH 7.4) at 4 °C for more than 2 days, embedded in Tissue Tek OCT compound (Sakura Finetek Japan, Tokyo, Japan), and rapidly frozen. Frozen frontal and horizontal sections (8-μm thickness) were prepared using a cryostat (Tissue-tek Polar DM; Sakura Finetek Japan) and mounted on slide glasses (Matsunami, Osaka, Japan). Nuclei were counterstained by incubation with Vectashield Mounting Medium containing 4,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA, USA). Sections were scanned using a fluorescence microscope (cellSens Dimension System; Olympus, Tokyo, Japan). Each section was scanned at least three times.

Water-soluble progesterone (PG), anesthetic MS-222 and ATP Bioluminescence Assay Kit CLS II were purchased from Sigma (St. Louis, MO, USA). hCG was from Teikoku Zoki (Tokyo, Japan) and collagenase (280 U/mg) was obtained from Wako (Osaka, Japan). The hydrogen peroxide colorimetric/fluorometric assay kit was from BioVision (Milpitas, CA, USA). Fluorogenic caspase-3 substrate IV was purchased from Calbiochem (La Jolla, CA, USA). Polyclonal anti-cyclin B2 antibody was ordered from Santa Cruz (Santa Cruz Biotechnology, Dallas, TX, USA), biotinylated anti-rabbit IgG was from Vector Laboratories (Burlingame, CA, USA). The Streptavidin Biotin Complex Peroxidase Kit, protein assay CBB and hydrogen peroxide were from Nacalai Tesque (Kyoto, Japan). MitoTracker Deep Red FM was from ThermoFisher (Waltham, MA, USA). Luciferase control RNA and luciferase assay system were from Promega (Madison, WI, USA). Other chemicals were obtained from Wako and Nacalai Tesque. Slide glasses and cover slips for microscopy were purchased from Matsunami Glass (Osaka, Japan).

After treatments, the tissues were transferred to Tissue-Tek optimal cutting temperature (O.C.T, Sakura Finetek, Tokyo, Japan) compound to snap freeze for immunohistology. IHC was done as previously described [32 (link),34 (link)]. Briefly, 10 µm tissue sections were air-dried on the slide glasses (Matsunami Glass, Osaka, Japan) for 40 min at RT, then washed with tris-buffered saline with 0.1% Tween20 (1× TBST) for 10 min. Tissues were blocked with 3% bovine serum albumin (BSA Gemini, Bio-Products, West Sacramento, CA, U.S.A.)/1× TBST for 1 h followed by overnight incubation with vimentin diluted at 1:300 (Abcam, Cat. No.: ab92547, Lot No.: GR3186827-13) and cytokeratin-18 diluted at 1:800 (Abcam, Cat. No: ab668, Lot No: GR3196069-6) in 3% BSA/1× TBST at 4°C. The next day, tissues were washed 3 times with 1× TBST for 10 min each, then treated with secondary antibodies diluted at 1:1000 (Alexa Fluor® 488, Abcam, Cat. No.: ab150073, Lot: GR269274-4 and Alexa Fluor™ 594, Invitrogen, Cat. No.: A11005, Lot: 2179228, respectively) in 3% BSA/1× TBST for 3 h at RT. Then, tissues were stained with NucBlue™ Fixed Cell ReadyProbes™ (DAPI, Invitrogen, Cat. No: R37606, Lot No: 2216969) for 5 min, and washed 3 times with 1× TBST for 10 min each. The images were taken with a Keyence microscope (Keyence Corp., Osaka, Japan).