Reelin

For immunohistochemistry, livers were fixed with formalin for 16 hours directly after harvest. Sections, 4 μm, were dewaxed and rehydrated, followed by antigen retrieval in boiling sodium citrate. Avidin and biotin were blocked according to the manufacturer's protocol (Vector, Peterborough, UK). Protein block (Dako, Cambridge, UK) or serum was added, and sections were then incubated with alpha‐smooth muscle actin (αSMA; Sigma‐Aldrich, Dorset, UK), collagen I, or GR1 (Ly6G) (Cambridge Bioscience, Cambridge, UK), F4/80 (Abcam, Cambridge, UK), or reelin (Abcam) antibodies at 4^oC overnight and subsequent secondary antibody for 30 minutes. After ABC vector incubation, sections were incubated with 3,3'‐diaminobenzidine for 10 minutes and counterstained with hematoxylin. Picrosirius red (PSR) staining was performed as described.3, 23 Thirty to 40 high‐power fields (magnification ×80) per section were randomly selected for each slide by an assessor blind to genotype. Images were analyzed in Photoshop CS3 for positively stained pixels and normalized to the total number of pixels. In the period acid–Schiff stain, the necrotic cell area was quantified in a blinded manner using the Image J Trainable Weka Segmentation tool.

Cells were fixed with 4% paraformaldehyde (Sigma), followed by membrane permeabilization and blocking with 0.2% Triton X-100 (Sigma) and 2% donkey serum (Jackson ImmunoResearch Laboratories). All quantifications were performed blinded to genotype in ImageJ. Antibodies: Nestin (R&D MAB1259), TBR2 (Abcam ab23345), PAX6 (Covance PRB-278P), BRN2 (Abcam Ab137469), PKCλ (BD Transduction Laboratory 610207), Cleaved caspase 3 (R&D Systems AF835), TUNEL (Sigma 12156792910), Acetylated alpha-tubulin (Cell Signaling 5335S), P73 (Abcam Ab40658), and Reelin (Abcam AB5364).