Anti cd204 clone sra e5

Paraffin sections were subjected to single and double IHC using a routine protocol that has previously been published [23 (link),24 (link),25 (link)]. In brief, anti-SPP1 antibody (AF1433; R&D Systems, Minneapolis, MN, USA), anti-Iba-1 antibody (Wako, Tokyo, Japan), anti-CD204 (clone SRA-E5; CosmoBio, Tokyo, Japan), and anti-PU.1 (clone EPR3158Y; Abcam, Cambridge, UK) were used as the primary antibodies. Anti-PU.1 is used as a pan-macrophage marker and it is positive in the nucleus [26 (link)]. All immunostained sections were evaluated by two investigators (Y.K. and E.M.). The SPP1 expression level on cancer cells and macrophages was scored according to the proportion of stained cells as follows: less than 1% staining, score 0; 1% to 49% staining, score 1; more than 50% staining, score 2.

The cellular proteins were solubilized in a Tris buffer containing 2% sodium dodecyl sulfate and 10% glycerol. The following antibodies were used for Western blotting: anti-SPP1 (AF1433; R&D Systems), anti-CD204 (clone SRA-E5; CosmoBio), anti-Bcl-2 (D17C4; Cell Signaling Technology, Danvers, MA, USA), anti-Bax (D2E11; Cell Signaling Technology), anti-cleaved caspase-3 (D175; Cell Signaling Technology), anti-cleaved PARP (Asp214; D64E10; Cell Signaling Technology), anti-Bmi1 (EPR3745(2); abcam, Cambridge, UK), and β-actin (sc-47778; Santa Cruz Biotechnology, Dallas, TX, USA). The detected bands were quantitatively measured using Image J [28 (link)]. The original WB can be found in the Supplementary Material File S1.