Ab200999

To determine the immune complex deposition in the kidney, paraffin-embedded renal sections were incubated with anti-mouse C3 antibody (Abcam, catalog ab200999) for mouse C3 staining and anti-mouse IgG antibody (Abcam, catalog ab205724) for mouse IgG staining. Fluorescence was labeled using Opal 7-color Manual IHC Kit (Perkin Elmer, catalog NEL811001KT). The image was captured by Perkin Elmer and analyzed by the Mantra system. The deposition of C3 and IgG was evaluated by Mean Fluorescence Intensity (MFI) using Fiji software (16 (link)).

Cells or lung tissue samples were lysed with RIPA buffer containing protease inhibitors and protein concentrations determined by BCA Protein Assay Kit (Thermo Fisher Scientific, 23227). Denatured proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membrane (Merck Millipore, IPVH00010). Blottings were incubated with primary Abs overnight at 4 °C and then with secondary Abs for 1 h at room temperature. The following primary antibodies were used: Mouse monoclonal anti-β-actin antibody (Sigma, A5441) (1 : 1000), Rabbit anti-C3 (Abcam ab200999) (1 : 500), Rabbit polyclonal to Histone H3 antibody (citrulline R2 + R8 + R17)-ChIP Grade (Abcam, ab5103) (1 : 500), and Rabbit anti-pSTAT6 (Cell Signaling Technology 56554) (1 : 500).

Renal tissues and intestinal tissues (sampled at the ileum and colon) were fixed with paraformaldehyde and embedded with paraffin. The sections were stained with H&E for observing the histological features. The renal tissues were also stained with recombinant anti-C3 antibody [EPR19394] (abcam, ab200999) or goat anti-mouse IgG antibody conjugated with Alexa Fluor 488 (abcam, ab150113) by immunofluorescence for observing the immune complex depositions in the kidney. The intestinal tissues were stained with the anti-ZO1 tight junction antibody (abcam, ab96587).

Four-micrometre tissue sections from formalin-fixed, paraffin-wax-embedded human endometriosis tissues and endometrial tissues were dewaxed, rehydrated, and subjected to high-temperature antigen retrieval. The tissues were incubated with blocking antibody diluent at room temperature for 2 h, and then incubated overnight at 4 °C with the following primary antibodies: anti-vimentin (mouse, ab8978, dilution 1:1000; Abcam), anti-CXCL8 (rabbit, sc-8427, dilution 1:150; Santa Cruz), anti-C7 (rabbit, ab126786, dilution 1:150; Abcam), anti-S100A10 (rabbit, sc-81153, dilution 1:150; Santa Cruz), anti-C3 (rabbit, ab200999, dilution 1:150; Abcam), and anti-StAR (rabbit, sc-166821, dilution 1:150; Santa Cruz). The slides were then incubated with secondary antibody (HRP polymer, anti-mouse/rabbit IgG, Abcam) at room temperature for 1 h. Nuclei were stained with 4-6-diamidino-2-phenylindole (DAPI, ab104139, Abcam) after all antigens had been labelled and then imaged.

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).

Immunofluorescence was performed to calculate the number of positive cells and to evaluate the activation states of microglia and astrocytes. Sections were rinsed three times in phosphate-buffered saline (PBS) for 5 min each at room temperature and then blocked with 1% donkey serum containing 0.3% Triton X-100 for 30 min. The sections were then incubated with primary antibodies overnight at 4 °C and with appropriate secondary antibodies at 37 °C for 2 h the next day. Finally, to label the nuclei, the sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). The following primary antibodies were used: anti-NeuN (1:100, Abcam, ab177487), anti-glial fibrillary acidic protein (GFAP) (1:400, ab4674, Abcam), anti-ionized calcium binding adapter molecule 1 (Iba1) (1:500, Abcam, ab178846), anti-iNOS (1:500, Abcam, ab49999), anti-C3 (1:300, Abcam, ab200999), and anti-Lcn2 (1:200, Abcam, ab63929). Images were obtained under a fluorescence microscope (BX51, Olympus).