iDISCO+ Clearing and Immunolabeling of Temporal Bone

For all experiments except blood vessel tracing, animals were anesthetized with diethyl ether until complete areflexia and transcardially perfused with heparinized Krebs solution until complete blood clearance, followed by 4% PFA in PBS. For blood vessel tracing, animals were anesthetized and decapitated without transcardiac perfusion; the brain was rapidly dissected out (3–6 min from decapitation to fixation) and immersed in 4% PFA. All samples were postfixed overnight in 4% PFA and then cryoprotected in 30% sucrose solution until sinking.
Cryoprotected samples containing the intact temporal bone were decalcified with buffered 10% EDTA in PBS (daily changed) until softening of the squamous temporal bone (3–4 weeks). Decalcified samples were embedded in 5% gelatin, trimmed, and halved. After trimming, gelatin was mechanically removed from the sample and clearing was performed using the iDISCO+ protocol (Renier et al., 2016 (link)). Samples (shown in Supplementary Figure S1 before and after clearing) were immunolabeled using rabbit anti-Iba1 (WAKO, 1:200) for macrophages/microglia, sheep anti-transthyretin (TTR; Abcam, 1:250) for CP epithelium and goat anti-rat IgG (Thermo Fisher, 1:200) for blood vessel lumen (as in, Liebmann et al., 2016 (link)). Species-matched Alexa-conjugated donkey secondary antibodies (Life Technology) were used at 1:200.