Supplementary Materials and Methods
Animals and Injections
All experiments involving animals were performed according to the ethical approval (AZ8.87-51.04.20.09.325) according to the declaration of Helsinki and its later amendments. Adult male C57BL/6N mice (Charles River, Cologne, Germany) were exposed to carbon tetrachloride (CCl4) for 6weeks (1ml/kg body weight (BW) mixed with olive oil (Rewe, Cologne, Germany) to 50μl; 3 injections per week i.p. according to Nussler et al. (2014)
Sirius red staining
Fibrotic scarring was visulaized by collagen deposition (Sirius Red staining) in liver tissue of mice as described (Krizhanovsky et al., 2008). Briefly, FFPE sections were deparafinized and rehydrated using xylene (Carl-Roth, cat. no. 9713.2) and ethanol (Carl-Roth, cat. no. K928.4) gradients. After brief washing under tap water, the sections were incubated with Sirius red (Direct red 80; Sigma-Aldrich Corp., C.I. 35782) for 30 minutes. Subsequently, the sections were washed with tap water and dehydrated with ethanol. Then the slides were mounted using malinol. Microscopy was performed with LEICA (Leika Mikroskopie and Systeme GmbH, Bensheim, Germany).
Confocal Microscopy
Liver slices, co-immunofluoscence staining as well as confocal scanning were generated as previousely described Reif et al. (2015) and Vartak et al. (2016). Breifly, immediately before staining, the liver lobe was sliced using a vibrating blade microtome (Vibratome VT1000 S, Leica, Wetzlar, Germany) into 75–100 μm thickness. After washing in 1XPBS, antigens were demasked using citrate buffer. Then, unspecific bindings were blocked by bovine serum albumin (BSA, Serva Electrophoresis GmbH, Heidelberg, Germany, 11926, 100 g) for 2h at room temperature. Subsequently, the slices were incubated with the first antibodies (Supplemental Table 2) overnight at 4°C. After 3 washing steps in 1XPBS, the slices were incubated with the second antibodies (Supplemental Table 2) overnight at 4°C. We used the second antibody DMs (Donkey anti-mouse IgG) to visualize the sinusoidal system and collagen depsotion. Justification of this staining was thoroughly described by Hammad et al. 2014. Counterstaining was performed using DAPI (4,6-diamidin-2-phenylindol, (Invitrogen, Darmstadt, Germany, D3571, 1:10.000) for 90min ar room temperature. Images were captured with LEICA confocal microscopy (Leika Mikroskopie and Systeme GmbH, Bensheim, Germany).
Supplementary References
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