Primary renal, spleen, and renal allograft tissue cross-sections were fixed in 10% formalin and embedded in paraffin. Five μm sections were cut with a Leica CM1900 cryomicrotome (Leica Biosystems Division of Leica Microsystems Inc.). The sections underwent H&E, PAS, and Masson's staining for histological evaluation. Immunochemistry staining was performed following the manufacturer’s protocol. Briefly, sections were deparaffinized by successively washing them with xylene, 100% and 95% ethanol, and distilled water. After antigen unmasking and blocking, the sections were incubated with anti-C4d antibody (HP8033; Hycult Biotech, USA) or anti-IL21R antibody (ab5980; Abcam, UK) overnight at 4°C. The rabbit-specific HRP/AEC IHC Detection Kit Micro-polymer (Abcam, UK) was used to visualize the stained sections, followed by staining with diaminobenzidine and counterstaining with hematoxylin. The acute humoral rejection was confirmed by the presence of DSA, linear C4d staining in peritubular capillaries, and histologic evidence of graft injury [44 (link)]. A well-experienced pathologist scored the histopathologic characteristics of rejection, including tubulitis, glomerulitis, interstitial inflammation, intimal arteritis, and peritubular capillaritis according to the Banff lesion scores [45 (link)]. ImageJ software was used to analyze the captured images.
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