α sma 1a4

Liver tissues were fixed overnight in 10% formaldehyde and were embedded in paraffin. Slides were stained with H&E, Masson's Trichrome, or Picric acid-Sirius red. For immunohistochemical analysis, paraffin-embedded or frozen sections were incubated with antibodies against mouse CD45 (30-F11; BD Bioscience), CD68 (KP1; Abcam), MPO (Rabbit polyclonal; Abcam), Dectin-1 (R1-8g7; Invivogen), PCNA (PC10; Biolegend), TLR4 (Rabbit polyclonal; Abcam), α-SMA (1A4; Abcam), Phalloidin (Cell Signaling), or M-CSF (Rabbit polyclonal; Abcam). Human liver sections were stained with an antibody against Dectin-1 (Rabbit polyclonal; Abcam) or CD11b (M1/70; Biolegend). Quantification was performed by examining 10 high powered fields (HPFs) per slide. Fibrosis was quantified based on Trichrome staining using a computerized grid as described (Ochi et al., 2012a (link)). Immunofluorescent imaging was performed using a LSM 700 confocal microscope and an Axiovert camera (Zeiss).

Rabbit explants (42 d) were stained for macrophage (MAC387; Abcam) and T-cell (CD3; courtesy of Dr. Peter Moore) presence throughout the scaffold and adjacent tissue. Pig explants (28 d) were stained for T-cell (CD3, SP7; Abcam), B-cell (CD79a, HM47/A9; Abcam), M1 macrophage (CCR7, C-terminal; Abcam), M2 macrophage (CD163; Abcam), endothelial cell (CD31; Abcam), and smooth muscle cell (αSMA, 1A4; Abcam) presence throughout the scaffold and adjacent tissue. Ratio of M2 to M1 macrophages was quantified from HPFs using NIS Elements at the implant middle and end (n = 2 HPFs per sample, n = 3 per group) to determine whether the implant was pro-inflammatory (M1 > M2) or pro-regenerative (M1 < M2).