Bz x710 inverted fluorescence microscope

Sections were cut to 4 μm thickness and placed on frosted histology glass slides (Thermo Fisher). H&E stained sections were obtained from each FFPE block. Deparaffinization, rehydration, and HIER were performed on an ST4020 small linear stainer (Leica) as described above. Nonspecific binding was blocked for 1 h at room temperature using 100 μL of serum-free protein block (Agilent). Antibodies were diluted in 100 μL antibody diluent (Agilent), and sections were stained overnight in a sealed humidity chamber at 4°C on a shaker. After staining, slides were washed for 10 min in 1x TBS IHC wash buffer with Tween® 20 (Cell Marque), and specimens were covered with dual endogenous enzyme-blocking reagent (Agilent) for 5-10 min at room temperature, followed by washing for 10 min. Bound antibodies were then visualized using the HRP/liquid DAB+ substrate chromogen system (Agilent) according to the manufacturer’s instructions. Sections were counterstained with hematoxylin, followed by dehydration, mounting, and imaging in brightfield mode on a BZ-X710 inverted fluorescence microscope (Keyence).

Under the same conditions described in Section 2.4, the MC3T3-E1 cells were seeded and cultured in µ-Slide 8 wells (ibidi GmbH, Martinsried, Germany). The cells were rinsed twice with DPBS after 24 h of NFP exposure to NFPs in MC3T3-E1 cells (NFP-MC) and stained with nuclear dye (H33342 fluorescent dye trihydrochloride in dimethyl sulfoxide solution; Nacalai Tesque, Kyoto, Japan) and lysosome staining solution (CytoPainter Lysosomal Staining ab138895; Abcam, Cambridge, UK) for 30 min [26 (link)]. Subsequently, the cells were washed twice with DPBS, and images were captured using a BZ-X710 inverted fluorescence microscope (Keyence, Osaka, Japan).
Furthermore, after incubation under the aforementioned conditions for 24 h, the cells were trypsinized, fixed using a 4% paraformaldehyde phosphate-buffered solution (4% PFA; Nacalai Tesque), centrifuged at 300× g for 10 min at room temperature, and the 4% paraformaldehyde phosphate-buffered solution was subsequently removed. The cells were resuspended in DPBS, and the side scatter light (SSC) was evaluated using a BD FACSCelestaTM flow cytometer (FCM; BD, Franklin Lakes, NJ, USA) [27 (link),28 (link),29 (link)].

Lungs were inflation fixed with 10% buffered formalin at 25 cm H₂0 pressure and fixed at 4°C overnight. Lungs were then processed and paraffin embedded, and 5 mm sections were used for all histological stains. Immunofluorescence was performed using antibodies (described in Supplemental Table 1) as follows. Antigen retrieval was done using a rice cooker and 1× Citrate Buffer (pH 6, Sigma Aldrich, C9999); slides were then washed in deionized water. Slides were blocked in 5% BSA for 1 hour at room temperature and then incubated in primary antibodies overnight. Samples were then washed 3× in PBST and then incubated in secondary antibody with DAPI diluted in PBST for 2 hours. Slides were then washed in PBST 3×, and cover slips were added with mounting media. For immunofluorescence analysis, samples were imaged in a Keyence BZ-X710 inverted fluorescence microscope, and images were taken on a 20× objective with 5–6 nonoverlapping fields of view. Image analysis was performed with HALO image analysis software for automated quantification of respective cell states. Histological analysis of lung injury was quantified using ImageJ (NIH) on samples stained with Masson’s trichrome stain, using a grid placed over the image assessing points at intersecting lines for lung injury (18 (link)).