Ab11878

Tissue microarrays (TMA) were established as previously described31 (link). Primary monoclonal anti-C5a antibody (1:100 dilution, ab11878, Abcam, Cambridge, MA, USA) were applied in the procedure. Three independent shots of C5a staining areas (away from tissue margin, obvious inflammatory and necrotic domains) were taken for analysis. The intensity was analyzed by Image-Pro Plus 6.0 and integrated optical intensity (IOD), representing the staining intensity, was recorded. The TMA slides were observed and evaluated by two investigators unaware of the clinical information. To determine the optimum cutoff IOD score, we performed X tile plot analysis with X tile software 3.6.1, and select the cut point by the rule of “minimum P value”. The cutoff point was 12578 for tumoral C5a staining, but not a significant point was found in peritumoral C5a staining.

IHC method was employed to detect regional deposition of complement components, including C3 (ab200999, Abcam), C3a (neo-epitope, ab11873, Abcam), C5a (neo-epitope, ab11878, Abcam), fB (ab192577, Abcam), and CR1 (anti-CD35, ab25, Abcam). IHC results were analyzed by two experienced pathologists blinded to patient’s information, and were scored by a semi-quantitative method in which staining of more than 10% of tumor cells was considered positive. The staining intensity was scaled as 0 for negative, 1 for weak (10~40%), 2 for moderate (40~70%) and 3 for strong (> 70%). The average score of staining intensity was calculated with five independent high-power fields using IMAGE PLUS software (Version 6.0, Media Cybernetics, USA). Low and high C3 deposition were defined as ≤1 and > 1 point, respectively. Deparaffinized sections from harvested human tissues were pretreated with 10 mM sodium citrate buffer (pH 6.0, boiling temperature, 30 min), blocked in normal serum (Vectastain ABC Kit; Vector Lab., Inc., CA, USA), incubated with primary antibodies (solution with saline, 1:100) at 4 °C overnight, rinsed and incubated with secondary antibody (EliVision plus, DAB Kit, 9902).