Blockade of the C5a–C5aR axis alleviates lung damage in hDPP4-transgenic mice infected with MERS-CoV

Jiang, Yuting; Zhao, Guangyu; Song, Nianping; Li, Pei; Chen, Yuehong; Guo, Yan; Li, Junfeng; Du, Lanying; Jiang, Shibo; Guo, Renfeng; Sun, Shihui; Zhou, Yusen

Sections of paraffin-embedded spleen and lung tissues (4 μm in thickness) were prepared to detect antigen expression through immunohistochemistry (IHC) staining. Briefly, the retrieved sections were incubated overnight at 4°C with the following antibodies: a mouse anti-C3 mAb (Hycult Biotech, The Netherlands), rabbit anti-C5b-9 (Calbiochem), and anti-C5aR (Santa Cruz Biotechnology, Paso Robles, CA, USA) polyclonal antibodies, rabbit anti-CD68 (Abcam, Cambridge, MA, USA) and anti-IFN-γRα (Santa Cruz Biotechnology) polyclonal antibodies, a rabbit cleaved caspase-3 Ab (Cell Signaling), a mouse anti-PCNA mAb (Santa Cruz Biotechnology), and a rabbit Novel coronavirus nucleoprotein/NP polyclonal Ab (Sino Biological Inc., Beijing, China). Biotinylated immunoglobulin G was then added, followed by an avidin-biotin-peroxidase conjugate (Beijing Zhongshan Biotechnology Co., Ltd.). Immunoreactivity was detected using 3,3' diamino benzidine (DAB) and by counterstaining with hematoxylin.