Table 2:

Reagents required for antibody labeling

Process	Solution	Duration (for 1-2 mm slices)	Duration (for hemibrain or whole brain)
Washing (PBST)	0.1 % Triton X-100 in PBS	1 hour (3 times)	1 hour (3 times)
Permeabilization (PBST-20)	0.2 % Tween-20 in PBS	1 hour	1 hour
Blocking	0.2% Triton-X-100, 10% DMSO, 6% Normal Donkey Serum in PBS	1 day	1 day
Primary antibody	0.2 % Triton X-100, 10% Normal Donkey Serum in PBS	2-3 days	1 week
Washing	0.1 % Triton X-100 in PBS	1 hour (3 times)	1 hour (3 times)
Secondary antibody	0.2 % Triton X-100, 10X Normal Donkey Serum in PBS	2-3 days	1 week
Washing	0.1 % Triton X-100 in PBS	1 hour (3 times)	1 hour (3 times)
TDE clearing (TDE I)	20% TDE for 1-2 mm slices, 30% TDE for hemibrain or whole brain	1-2 hours for 1-2 mm slices,	24 hours for hemibrain or whole-brain*
Refractive index matching (TDE II)	47% TDE for 1-2 mm slices, 63% TDE for hemibrain or whole brain	2 hours for 1-2 mm slices	24-48 hours for hemibrain or whole-brain*

^*hemibrain or whole brain should be imaged by using light-sheet microscopy. 1-2 mm slices can be imaged using confocal or multiphoton microscopy.