Ab55445

Tissue microarray blocks were constructed. A core (3 mm in diameter) of representative invasive tumor area was selected from each case. Tissue sections were stained with monoclonal anti‐MIF antibody at a dilution of 1:100 (Abcam, ab55445), using the automated immunostainer (Benchmark Ultra, Ventana Medical Systems Inc., Tucson, AZ, USA). The positive control was using tumor‐infiltrating macrophages in SCC of the lung. The primary antibody was omitted for the negative control. The representative images are shown in Figure 1a,b.
MIF expression was assessed according to the widely‐used Remmele system.12 The intensity of staining of tumor cells was scored as 0 (no color reaction), one (mild reaction), two (moderate reaction) and three (intense reaction). The percentage of positive tumor cells was classified as <10%, 10%–50%, 51%–80% and >80% positive cells. In this study, each case was subdivided as low (<2 or <80%) or high (others) group based on the intensity score and percentage of positive tumor cells, respectively.

Representative hematoxylin and eosin-stained glass slides containing tumoral lesion specimen from the 152 CCRCC patients were reviewed and selected by the 2 pathologists. Two 3-mm cores were obtained from each representative intratumoral lesion in the paraffin block and transplanted to recipient tissue microarray (TMA) blocks. Immunohistochemical staining was performed on 4-μm sections of the TMA block samples. The sections were attached to glass slides, deparaffinized, rehydrated, and incubated in 3% hydrogen peroxide for 10 minutes to block endogenous peroxidase activity. Each section was heated for 20 minutes in 10 mM citrate buffer (pH 6.0) in a microwave oven (700 W). After incubation with Ultra V block (LabVision Corporation, Fremont, CA, USA) for 7 minutes at room temperature to block background staining, the slides were incubated with an MIF monoclonal primary antibody (1:1000 dilution, ab55445, Abcam, Cambridge, United Kingdom). An ultraView Universal DAB detection kit was used (760–500, Ventana, Tucson, AZ, USA) according to the manufacturers recommendation. 3, 3′-Diaminobenzidine was used to detect the protein reactivity. The sections were counterstained using hematoxylin.

Proteins were extracted using RIPA lysis buffer (Thermo Fisher Scientific, #89900, MA, USA) containing protease inhibitor cocktail (Thermo Fisher Scientific, #78430, MA, USA). The total protein concentration of each cell lysate was measured by the Bradford method using bovine serum albumin as a standard. Equal amounts of protein lysates (45 μg) were loaded on a denaturing polyacrylamide gel and then transferred to a nitrocellulose membrane. The primary antibodies used for immunoblotting were anti-MIF (ab55445, Abcam, Cambridge, United Kingdom), and anti-GAPDH (ab8245, Abcam, Cambridge, United Kingdom) antibodies. Subsequently, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies and developed by an enhanced chemiluminescence reaction (Thermo Fisher Scientific, #32109, MA, USA). Digital chemiluminescence images were captured with a Fusion Solo instrument (Vilber, Collégien, France). The software Evolution Capt was used to quantify the gray value of the bands from the western blot.