Optimized CLARITY procedures for faster, simpler and affordable whole brain clearing and imaging
The three key features of this new approach were: 1) accelerated clarification through parallelized flow-assisted clearing crucial for large cohorts independent of specialized equipment such as electrophoresis or perfusion chambers; 2) >90% cost reduction (also important for these large behavioral cohorts) using a new refractive index-matching process; and 3) optical properties such that the whole mouse brain can be imaged using a commercial light-sheet microscope (LSM) under a single field of view (FOV) and as a single stack (~1200 steps across a ~6.6mm range) in less than 2 hours with single-cell resolution throughout the whole volume (this speed and simplicity is also critical for large behavioral cohorts). Raw data files from each brain are ~12 GB in size and can be easily stored and directly analyzed on standard desktop workstations without the need for compression or stitching.
A hydrogel based on 1% acrylamide (1% acrylamide, 0.0125% Bis, 4% PFA, 0.25% VA-044 initiator (w/v), in 1X PBS, as described in Tomer et al 2014) was used for all CLARITY preparations. Mice were transcardially perfused with ice-cold 4% PFA (not hydrogel yet). After perfusion, brains were post-fixed in 4% PFA overnight at 4°C and then transferred to 1% hydrogel for at least 48 hours to allow monomer diffusion. The samples were degassed and polymerized (4-5 hours at 37°C) in a 50ml tube. The brains were removed from hydrogel and washed with 200mM NaOH-Boric buffer (pH=8.5) containing 8% SDS for 6-12 hours to remove residual PFA and monomers. Brains could now be transferred to a flow-assisted clearing device using a temperature-control circulator or a simper combination of 50ml tube and heated stirring plate. 100mM Tris-Boric Buffer (pH=8.5) containing 8% SDS was used to accelerate the clearing (at 40°C). Note that Tris-containing buffer should only be used after PFA is completely washed out. With this setup, a whole mouse brain can be cleared in 12 days (with circulator, or 8 days for a hemisphere) or 16 days (with conical tube/stir bar). After clearing, the brain was washed in PBST (0.2% Triton-X100) for at least 24 hours at 37°C to remove residual SDS. Brains were incubated in a refractive index matching solution (RapidClear, RI=1.45, Sunjin lab, http://www.sunjinlab.com/) for 8-12 hours at 37°C and then 6-8 hours at room temperature. Sample can be stored in RC for several days at room temperature without affecting imaging quality. After the RC incubation, the brains were ready for imaging.