CUBIC Tissue Clearing and Immunostaining

CUBIC clearing was based on the modified version of the protocol by Susaki et al.^{37 (link)}, that utilizes the CUBIC R1a method (unpublished, protocol available at http://cubic.riken.jp/) as previously described by Lloyd-Lewis et al.^{38 (link)}. Briefly, fixed samples were placed in R1a for 3 days in a dry incubator at 37 °C, changing to fresh R1a each day. Samples were blocked overnight in blocking buffer (10% normal goat serum in PBS-0.5% Triton-X). Primary antibodies were diluted in blocking buffer for 4 days at 4 °C in the following concentrations: rabbit anti-perilipin 1:50 (Cell Signalling, D418), rabbit anti-collagen IV 1:100 (Abcam, ab6586), rabbit anti-laminin 1:100 (Abcam, ab11575), rabbit anti-HER2 1:100 (Dako, A0485) and the nuclear dye SYTO16 at 1 µM (Life technologies, S7578). Secondary antibody Alexa Fluor 647 goat anti-rabbit (Life Technologies, A21245) was diluted 1:500 and incubated for 2 days at 4 °C.