Anti cd74

Radio‐immune precipitation assay lysis buffer (Beyotime, P0013B) was used for tissue and cell lysates. BCA assay kit (Beyotime, P0012S) was used for protein concentration determination. After 30 µg protein amount was separated using 4%–20% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, it was transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% skim milk at 37°C for 2 h and then incubated with primary antibodies at 4°C overnight and secondary antibodies at 37°C for 1 h. After washing, the membranes were visualized by an ECL Kit (MedChemExpres) and quantified with ImageJ software. All experiments were repeated in triplicate for statistics. Following primary antibodies were used: anti‐iNOS (1:1000, Proteintech), anti‐COX‐2 (1:1000, Proteintech), anti‐IL‐6 (1:1000, Proteintech), anti‐TLR4 (1:1000, Proteintech), anti‐MYD88 (1:1000, Proteintech), anti‐NF‐κB‐p65 (1:1000, Affinity), anti‐NF‐κB‐p‐p65 (1:1000, Affinity), anti‐CD74 (1:1000, Abcam), β‐Actin (1:2000, Abcam).

From block, 4-μm-thick slices were cut and dewaxed by successive immersion in pure xylene (Sigma). Rehydration and unmasking of antigens were carried out by sequentially immersion into 100%, 90%, 80% and 70% ethanol baths (Sigma), phosphate-buffered saline (PBS; Gibco), and citrate buffer (pH 7). Saturation of nonspecific antigenic sites was achieved by incubation in PBS containing 3% bovine serum albumin (BSA). The primary antibodies were diluted in 3% BSA and incubated overnight at 4°C. The antibodies used were as follows: anti-CD74 (1:50, mouse, Abcam), anti-CD90 (1:50, rabbit, Abcam), anti-CD3 (dilution recommended by the supplier, rabbit, Ventana). The sections were subsequently incubated with a secondary antibody coupled to Alexa Fluor 594 or 488 (Molecular Probes) for 60 min. After washing with PBS, nuclear counterstaining was carried out with 4′,6-diamidino- 2-phenylindole (Sigma) and the sections were mounted in an aqueous medium (Vectashield, Sigma). Images were acquired with Leica microscope (DMi8, Leica).