Nb600 408

For general histology, in paraffin sections from lung tissue samples, Hematoxylin and eosin (H&E) staining were performed according to standard steps.
For immunofluorescence, 6-μm sections were treated with xylene and graded alcohols. Antigen retrieval was accomplished by microwaving slides in EDTA antigen retrieval buffer (pH 8.0). The slides were blocked with 5% BSA for 1 h and then incubated overnight at 4°C with rabbit polyclonal anti-collagen-I antibody (1:50; NB600-408; NOVUS), mouse monoclonal anti-elastin antibody (1:50; NB100-2076; NOVUS), or mouse anti-Fibrinogen beta chain (FGB) antibody (1:50; M01204-1; Boster), followed by Alexa Fluor 488-conjugated goat anti-rabbit secondary antibodies (A-11008, Invitrogen) or Alexa Fluor 488-conjugated goat anti-mouse secondary antibodies (A32723, Invitrogen) for 1h at room temperature and nuclei were stained with DAPI (62248, Invitrogen). The Pannoramic 250 Flash III (3DHISTECH) was used to scan the sections, and CaseViewer 2.4 was used to record the images.

To determine the expression of COL1A1 (1:200; NB600-408; Novus Biologicals) (Jun et al., 2019 (link)) after uterine damage, frozen uterine sections (12 μm) were fixed in 4% paraformaldehyde in phosphate buffered saline (PBS) for 10 min at room temperature, washed in PBS, and treated with 0.1% Triton X-100 for 10 min at room temperature and washed in PBS. Non-specific staining was blocked using protein block serum (Dako, Carpinteria, CA, United States). The sections were then incubated overnight with primary antibody at 4°C, washed in PBS, and incubated with secondary antibody (A-11001, 1:1000; Invitrogen) for 60 min at room temperature. After three washes in PBS, the sections were counterstained and mounted. Images were obtained using a suitable microscope (Carl Zeiss, Oberkochen, Germany) and analyzed using ZEN software (Carl Zeiss).

RAW264. 7 or HGF-1 cells seeded in confocal dishes were fixed with 4% paraformaldehyde at 4°C for 15 minutes after washed by PBS 3 times for 20 minutes each time. The cells were then permeabilized with 0. 2% Triton X-100 for 10 minutes and blocked with 2% BSA for 2 hours, and each step was followed by twice PBS washes to remove residual reagents. After the above steps were completed, primary antibodies were added to the cells and incubated at 4°C overnight. The primary antibodies were added as follows: Piezo1 antibody and CD68 were co-incubated to localize Piezo1 on macrophages; CCR7 (1:200;NB100-712; Novus Biologicals, USA) and CD206 (1:200; #24595; Cell Signaling Technology, USA) were co-incubated to identify macrophage polarization; COL-I (1:200;NB600-408; Novus Biologicals, USA) or COL-III (1:200; NB600-594; Novus Biologicals, USA) was incubated to indicate the existence of collagen in HGF-1. Afterwards, the cells were incubated with donkey anti-goat secondary antibody IgG H&L Alexa Fluor 594 (1:200; ab150132; Abcam, UK) and the goat anti-rabbit IgG H&L Alexa Fluor 488 for one hour at room temperature in the dark. After washing away the secondary antibodies with PBS, DAPI was used to stain the nuclei for 10 minutes in the absence of light. Finally, the images were acquired by the confocal laser scanning microscope and quantitatively analyzed by Image J software.