Anti cd74

Tissue and cell samples were homogenized in lysis buffer, separated by 10% or 12% SDS-PAGE under reducing conditions and transferred to PVDF membranes (Millipore, Bedford, MA, USA), blocked with 5% skimmed milk in PBS/0.5% v/v Tween 20 for 1 h, and washed with PBS/Tween [28 (link)]. Primary antibodies were rabbit polyclonal anti-CD74 (1:500, Santa Cruz, CA, USA), anti-MIF (1:500, Santa Cruz, CA, USA) and anti-DDT (1:500, Abcam). Antibodies were diluted in 5% milk PBS/Tween. Blots were washed with PBS/Tween and subsequently incubated with appropriate horseradish peroxidase-conjugated secondary antibody (1:2000, GE Healthcare/Amersham, Aylesbury, UK). After washing, blots were developed with the chemiluminescence method (ECL). Blots were then re-probed with monoclonal anti- mouse α-tubulin antibody (1:2000, Sigma) and levels of expression were corrected for minor differences in loading.

Following treatment with 2 μg/mL MIF in the presence or absence of 50 μM 4-IPP for 24 h, primary joint capsule fibroblasts were fixed in 4% paraformaldehyde with 0.1% Triton X-100 (Sigma, St Louis, MO, USA) and subsequently incubated with 4% goat serum to block nonspecific binding. Then, cells were incubated with anti-TGF-β1 (Servicebio, Wuhan, China, 1:100), anti-MIF (Abcam, 1:100), or anti-CD74 (Santa Cruz, 1:50) primary antibodies overnight. Fluorescent secondary antibodies of FITC-labeled goat anti-rabbit IgG (Sigma, 1:400) and Cy3-labeled goat anti-rabbit IgG (Sigma, 1:400) were used to visualize the corresponding subsets. Cells were then stained with 4,6-diamidino-2-phenylindole (DAPI; Sigma, 1:4000) and phalloidin (Abcam, 1:1000), followed by observation under a confocal fluorescence microscope (Leica, Germany) and semi-quantitatively analyzed with Image-J software (National Institutes of Health, USA). The fluorescence was scored and quantified with blinding.

Immunohistochemistry was carried out as previously described on paraffin-embedded 5 μm thick tissue sections [27 (link)]. Primary antibodies were rabbit polyclonal anti-CD74 (1:50, Santa Cruz, CA, USA), anti-DDT (1:100, Abcam) and anti-MIF (1:100, Santa Cruz). Sections were counterstained with Carazzi`s hematoxylin. Negative controls included incubation with a non-specific immunoglobulin of the same isotype as the primary antibody. Sections were subsequently incubated with the proximal tubule marker, fluorescein-conjugated tetragonolobus lotus lectin (1:33, Vector Lab, Peterborough, United Kingdom). Staining was evaluated by a quantitative scoring system, Image-Pro Plus software (Media Cybernetics, Bethesda, MD) in 10 randomly selected fields (x20) per kidney. Samples were examined in a blinded manner.