P jak2 tyr1007

A quantity of 10 μg of protein was loaded onto a 10% gradient polyacrylamide gel, electrophoretically transferred to a polyvinylidene difluoride membrane and probed with the following primary antibodies: S100A8/A9 (1:1000, Abcam, UK), Col1 (1:2000, Proteintech, China), α-SMA (1:6000, Proteintech, China), JAK2 (1:1000, Proteintech, China), p-JAK2 (Tyr1007) (1:1000, Absin, China), STAT3 (1:2000, Proteintech, China), p-STAT3(Tyr705) (1:1000, Absin, China), RAGE(1:1000, Proteintech, China), TLR4 (1:1000, Proteintech, China),  and GAPDH (1:1000, Absin, China), which was used as an internal control. Secondary antibodies were horseradish peroxidase-conjugated to mouse anti-rabbit/mouse IgG (1:5000, Proteintech, China).

Immunohistochemistry was carried out by using S100A8/A9 (1:1000, Abcam, UK), Col1 antibody (1:1000, Proteintech, China), α-SMA (1:500, Proteintech, China), RAGE (1:200, Proteintech, China), p-JAK2(Tyr1007) (1:100, Absin, China), p-STAT3 (Tyr705) (1:100, Absin, China), CK18(1:1000, Proteintech, China), and vWF (1:1000, Proteintech, China). In brief, formalin-fixed, paraffin-embedded tissues were cut into 5-μm thickness and subjected to deparaffinization and rehydration. Following antigen retrieval, tissue sections were incubated with primary antibody overnight at 4 °C. After washed with PBS, sections were incubated with biotinylated secondary antibody for 1 h at room temperature. A DAB kit was employed as the chromogen and slides were counterstained with hematoxylin.
After deparaffinization, the sections were heated in EDTA for antigen retrieval and then blocked with BSA for 30 min at room temperature. The sections were then incubated with two kinds of primary antibodies at 4 °C overnight in a humidified chamber: CD16 antibody (1:5000, Servicebio, China) and S100A8/A9 antibody (1:5000, Abcam, UK). The next day, the sections were incubated with secondary antibodies for 50 min at room temperature. The secondary antibodies used were goat anti-rabbit Cy3 and goat anti-rabbit iF647 (both from Servicebio, China, diluted at 1:300). The nuclei were stained with DAPI for 10 min.