The PACT protocol is a derivative of the original CLARITY hydrogel-based clearing method. It was first introduced by Yang et al., 2014. Cell 158, 945-958. [doi:https://doi.org/10.1016/j.cell.2014.07.017]

Tissue-gel hybrid formation

1. Incubate fixed specimens incubated at 4°C overnight in the hydrogel monomer solution A4P0 (4% acrylamide in PBS) supplemented with 0.25% photoinitiator VA-044 [2,20-Azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride].

2. Incubate the A4P0-infused samples for 2–3 hr at 37°C to initiate tissue-hydrogel hybridization.

3. After removing excess hydrogel brief PBS washes, transfer the tissue-hydrogel matrix into a tube containing 8% SDS in PBS (pH 7.5), and incubate for 2 days at 37°C with shaking.

4. (optional) Performing immunostaining or other labeling after SDS treatment. Due to the slow penetration rate, all steps in the staining protocol need to be extended significantly.

RIMS

Mix 40 grams of Histodenz (Sigma D2158) with 30 ml of PBS, with 0.1% Tween-20 (and 0.01% sodium azide), pH to 7.5 with NaOH. Samples are incubated in RIMS until transparent.