Whole Mount Staining of Human Liver

Human liver tissue was fixed in 4% formaldehyde in phosphate buffered saline (PBS) for 2 h, permeabilized (5% Triton X-100/PBS) and subsequently blocked in Permblock solution (3% BSA, 0.5% Tween-20 in PBS). For whole mount immunostaining anti-cytokeratin 19 (proteintech, number 14965-1-AP), anti-αSMA-Cy3 (Sigma, clone 1A4) and Alexa647-coupled secondary antibody (Molecular Probes) were used in Permblock solution. Antibody incubation was performed for at least 3 weeks at 37°C and samples were washed with PBS-T (0.1% Tween-20/PBS) after each step. The whole mount stained samples were embedded in cylindrical 1% low melting agarose to avoid light scattering at agarose edges during later imaging. Following dehydration and delipidation in increasingly concentrated methanol (70%, 95%, > 99%, > 99%, each step at least 2 h), optical clearing was performed by gradually replacing methanol with a 1:2 benzyl alcohol-benzyl benzoate solution (BABB, Murray’s clear) for refractive index matching. Samples were equilibrated in BABB at least one day and subsequently imaged by light sheet fluorescence microscopy (LSFM) and optical projection tomography (OPT).