A5864

Samples were fixed with 4% paraformaldehyde overnight, then embedded in paraffin. 5 mm sections were processed and stained with hematoxylin and eosin (H&E) and Masson’s Trichrome Stain Kit following protocol (Solarbio, Beijing, China).
We used the following antibodies to investigate protein expression in these samples by immunohistochemistry: Piezo1 (ab128245, 1:100, Abcam, Cambridge, UK), CCL24 (22306-1-AP, 1:200, Proteintech, Wuhan, China), CCR3 (ab32512, 1:100, Abcam), Wnt2 (A5864, 1:200, Abclonal, Wuhan, China), Wnt11 (ab31962, 1:200, Abcam), α-SMA (#19245, 1:200, Cell Signaling Technology, Danvers, USA), p-JNK (sc-6254, 1:100, Santa Cruz Biotechnology, Dallas, Texas, USA), β-catenin(#8480, 1:100, CST). Images were captured using a Nikon Eclipse E800 microscope (Nikon, Melville, NY, USA).

Cells were lysed with ice-cold RIPA buffer and centrifuged at 13,000 g for 15 min at 4 °C. 10 μg of protein lysate (concentration determined by a bicinchoninic acid assay) was run on a 10% SDS-PAGE gel at 150 V for 1 h and then transferred to nitrocellulose filter membranes (Millipore, Bedford, MA). After blocking with 5% BSA at RT for 1 h, the membranes were incubated with the following primary antibodies: Piezo1 (ab128245, 1:1000, Abcam), Collagen I (ab34710, 1:500, Abcam), Collagen III (ab7778, 1:500, Abcam), fibronectin (ab2413, 1:1000, Abcam), α-SMA (#19245, 1:1000, CST), GAPDH (#5174, 1:1000, CST), CCR3 (ab32512, 1:1000, Abcam), Wnt2 (A5864, 1:1000, Abclonal), Wnt11 (bs-8568R, 1:1000, Bioss), JNK (#9252, 1:1000, CST), p-JNK (#4668, 1:1000, CST) at 4 °C overnight. The next day, membranes were incubated with an Anti-rabbit IgG, HRP-linked Antibody (7074 S, 1:1000, CST) at room temperature for 1 h and then washed with tris-buffered saline with 0.1% Tween 20 three times for 10 min. Quantitative analysis was performed on the immunoreactive bands with ImageJ software.

Cell samples were fixed with 4% PFA for 15 min at RT. Tissues were fixed with 4% PFA overnight at RT. Tissue samples were embedded in paraffin and sliced into 5 mm sections. Samples were incubated with following antibodies: Piezo1 (ab128245, 1:100, Abcam), α-SMA (ab7817, 1:100, Abcam), CCL24 (22306-1-AP, 1:200, Proteintech), CCR3 (ab32512, 1:100, Abcam), Wnt2 (A5864, 1:200, Abclonal, Wuhan, China), Wnt11 (ab31962, 1:200, Abcam), β-catenin(#8480, 1:100, CST), Alexa Fluor 594 goat anti-rabbit secondary antibody (Jackson lab, 125369, 1:200) and Alexa Fluor 488 goat anti-mouse secondary antibody (Jackson lab, 133384, 1:200). Images were visualized using a Nikon Eclipse E800 microscope (Nikon, Melville, NY, USA).