Akr1b1

Sections were deparaffinized, hydrated, and incubated in 3% hydrogen peroxide for 10 min, then washed three times for 3 minutes each in PBS (pH 7). Put the sections into sodium citrate buffer (pH 6.0), heated them in the microwave twice for ten minutes, and set them aside to cool at room temperature. and washed three times in PBS (pH 7.4) for 3 minutes each. According to the size of the tissue, drop an appropriate amount of primary antibody, incubate at 4°C overnight, washed three times for 3 minutes each in PBS. Antibodies used include STC1 (1:50; Proteintech, China), RPL5(1:800; Proteintech, China), AKR1B1(1:50; BOSTER, China), HLA-A(1:100; BOSTER, China) and CD47(1:400; BOSTER, China). Add an appropriate amount of enhanced enzyme-labeled goat anti-mouse/rabbit IgG polymer dropwise, incubate at room temperature for 20 minutes, washed three times for 3 minutes in PBS each. Use freshly prepared DAB chromogenic solution for visualization, rinse with PBS, and then counterstained with hematoxylin staining solution. The IHC results were viewed by an independent pathologist and whole section scanning was done using Pannoramic Scan (3DHISTECH, Budapest, Hungary).

Whole-cell protein was extracted using a protein lysis buffer(Beyotime, Shanghai, China).25μg samples were loaded in the well and were separated by 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). Then blocked with 5% nonfat milk powder in TBST (TBS with 0.1% Tween) for 2 hours at room temperature and incubated with the appropriate primary antibody at 4°C overnight. Antibodies used include AKR1B1(1:50; BOSTER, Wuhan, China) and β-tubulin(1:4000; Abmart, Shanghai, China). The next day these membranes were washed with TBST (TBS with 0.1% Tween) three times for 5 minutes each. Then membranes were incubated with HRP-coupled anti-rabbit secondary antibodies (1:1000; ZSGB-BIO, Beijing, China) and anti-mouse secondary antibodies (1:1000; ZSGB-BIO, Beijing, China) at room temperature for 2 hours. Next, membranes were washed with TBST (TBS with 0.1% Tween) three times for 5 minutes each. Finally use ECL reagents (Beyotime, Shanghai, China) and visualize by an enhanced chemiluminescence detection system (Thermo Scientific, Waltham, MA, USA).