Anti human ido1

Cells were collected, lysed in M2 lysis buffer and sonicated. The protein concentrations were determined by a BCA kit (Applygen Technologies Inc., China). Then, the protein was run on a SDS-PAGE gel and transferred to nitrocellulose. Nitrocellulose membranes were blocked in 5% bovine serum albumin (BSA) and probed with antibodies overnight: anti-actin (Cell Signaling, Cat. 3700; 1:1,000); anti-human IDO1 (Cell Signaling, Cat. 86630; 1:1,000); anti-mouse IDO1 (Merck, Cat. 05–840; 1:1,000); anti-mucin 1 (Abcam, Cat. ab45167; 1:1,000); anti-mucin 2 (Abcam, Cat. ab272692; 1:1,000); anti-mucin 5A (Abcam, Cat. ab24071; 1:100); anti-mucin 5B (Abcam, Cat. ab77995; 1:1,000); anti-mucin 6 (Abcam, Cat. ab192318; 1:1,000); anti-mucin 7 (Abcam, Cat. ab105466; 1:1,000); anti-mucin 13 (Abcam, Cat. ab124654; 1:1,000) or anti-mucin 16 (Abcam, Cat. ab1107; 1:1,000). Secondary antibodies conjugated to horseradish peroxidase were followed by enhanced chemiluminescence (Thermo fisher, MA). Results were confirmed by at least three independent experiments.

Tissue from ccRCC tumors and adjacent normal kidneys were archived following IRB approval at UC Davis Department of Pathology. Paraffin sections (4 μm) of formalin-fixed tissue were stained for IDO1 using heat-induced antigen retrieval in citrate buffer (pH 6.0) and anti-human IDO1 (Cell Signaling Technology), followed by Mach 2 Rabbit HRP-Polymer (BioCare Medical, Concord, CA) and ImmPACT diaminobenzidine peroxidase substrate (Vector Laboratories, Burlingame, CA). Immunohistochemistry for CD68 was done using heat-induced antigen retrieval at pH 9 (Dako, Carpinteria, CA), mouse monoclonal anti-CD68 (Leica Biosystems, Newcastle Upon Tyne, UK) and Mouse-on-canine HRP-polymer (BioCare Medical) prior to detection with NovaRed (Vector Laboratories). A pathologist (F.U.) scored IDO staining using a subjective scale of 0 (no IDO staining) to 3 (20-40% of cells were stained) without assigning a grade to the tumors in order to minimize possible bias of the IHC analysis. No more than 40% of all cells in any slide stained positive. Staining of CD68 was scored as a percentage of interstitial cells that were positive with a score 0 (no staining) to 5 (40-50% positive stained interstitial cells).