Ab209027

The paraffin-embedded tissue was cut into 8 μm-thick sections. After dewaxing and hydrating, immunostaining was conducted. Sections were pretreated with 0.1% Triton X-100 in PBS with 1% bovine serum albumin for 1 h, 1% t20 for 20 min, and then PBS for 20 min. Briefly, the sections were incubated for 30 min in the dark. The excess dye was washed away with PBS. Sections were incubated with isotype antibodies to exclude false positive staining. At least three parallel sections from different implantation sites were observed by fluorescence microscopy (Zeiss stereoscopic finding, V20, Germany). Five fields were randomly selected for immunofluorescence determination. Macrophages were magnified 400-fold to exclude false positive staining. Semiquantitative analysis was then performed at a magnification of 40-fold. CaseViewer 2.1 and Image Pro Plus 7.0 were used to measure the fluorescence intensity of five random spots. Hoechst (ab138903, Abcam) staining analysis was performed at the same time as CD68 (ab222914, Abcam) and inducible nitric oxide synthase (iNOS, ab209027, Abcam) analysis.

The antibodies used for immunohistochemistry staining and immunofluorescence staining were purchased from Abcam (Shanghai, China) including anti-TNF-α antibody (ab6671, 1:200), anti-IL-1β antibody (ab9722, 1:400), anti-IL-6 antibody (ab83339, 1:2000), anti-F4/80 antibody (ab6640, for immunohistochemistry staining, 1:200), anti-F4/80 antibody (Alexa Fluor 488, ab204266, for immunofluorescence staining, 1:400), anti-inducible nitric oxide synthase (iNOS) antibody (Alexa Fluor 647, ab209027, 1:200), anti-Mannose receptor antibody (anti-CD206, Alexa Fluor 647, ab195192, 1:200), anti-Galectin 3 (Mac-2) antibody (ab53082, 1:200).