Wnt5a antibody

Lung tissue was cut into 4 μm thick slices. Repair antigen under high pressure in sodium citrate (pH = 6.0). Wnt5a antibody (1 : 100; Abcam; Cambridge, MA, USA), β-catenin antibody (1 : 500; Abcam; Cambridge, MA, USA), and APC rabbit antibodies (1 : 100; Abcam; Cambridge, MA, USA) were incubated overnight at 4°C and negative controls were incubated with PBS. On the next day, horseradish peroxidase (HRP) labeled goat anti-rabbit IgG (Beijing Zhongshan Jinqiao Technology Co., Ltd.) was added dropwise. The incubation temperature was 37°C for 30 minutes and rinsed with PBS three times. Add 3,3′-diaminobenzidine (DAB) (Beijing Zhongshan Jinqiao Technology Co., Ltd.) for color development. Hematoxylin was counterstained and mounted. Observed under light microscope, the tissues were dark brown for protein positive expression. No coloration or light coloration is negative or weak positive expression. Finally, the average optical density (AOD) expressed by IPP software was analyzed. The integrated optical density (IOD)/positive expression area is AOD.

The tissue is quickly removed from the liquid nitrogen, and the lung tissue is ground into a powder. Collect lung tissue powder into a centrifuge tube, and add tissue lysate and pancreatin, respectively. The supernatant after centrifugation is a protein extract. BCA protein assay was used to determine protein concentration. Protein separation is done by polyacrylamide gel electrophoresis. The electrophoretic transfer method of wet transfer is used to transfer the protein to the PVDF membrane and block it in skim milk. Wnt5a antibody (1 : 500; Abcam; Cambridge, MA, USA), β-catenin antibody (1 : 5000; Abcam; Cambridge, MA, USA), APC antibody (1 : 500; Abcam; Cambridge, MA, USA), and GAPDH antibody (1 : 500; Abcam; Cambridge, MA, USA) were incubated overnight at 4°C. Wash the PVDF membrane 3 times with Tris-HCl buffered saline + Tween (TBST). A secondary antibody labeled with horseradish peroxidase (goat anti-rabbit IgG) was added for reaction. TBST washed the PVDF membrane 3 times again. Finally, with the help of ECL chemiluminescent reagents, the PVDF film shows the image. Analysis was done using Image J software.