*2.10. Immunostaining of Liver Slices*

CYP2E1 immunostaining was done in 200 μm-thick liver slices prepared using a cryostat microtome. After washing, permeabilization and blocking steps the slices were incubated for 3 days with a primary antibody against CYP2E1 (Sigma-Aldrich Corp., St. Louis, MO, USA, 1:50). Subsequently, the slices were incubated for 2 days with CyTM3-conjugated AffiniPure; F(ab')2 fragment donkey anti-rabbit secondary antibody (Dianova, Hamburg, Germany, 1:100). The nuclei were visualized by counterstaining with 4,6-diamidino-2-phenylindole. In order to allow 3D imaging of the thick slices, the tissue was cleared by successive immersion in 50% (*v*/*v*), 75% (*v*/*v*) and 100% (*v*/*v*) tetrahydrofuran, 15 min each, followed by immersion in di-benzyl ether (Sigma-Aldrich Corp., St. Louis, MO, USA) for at least 10 min. Subsequently, Z-stacks of approximately 120 μm-depth were acquired using a custom-modified inverted LSM MP7 (Zeiss, Jena, Germany) with an LD C-Apochromat 40 × 1.1 water immersion objective.
