Benzoic acid benzyl benzoate

Freshly isolated epididymal fat pads were cut into small pieces then fixed in 4% PFA/PBS at 4°C overnight. After permeabilization for 18 h in 0.3% (vol/vol) Triton X-100 PBS at 4°C, the samples were blocked with 5% (vol/vol) donkey serum in 0.05% (vol/vol) Triton X-100 PBS for 12 h at 4°C and then incubated with the primary antibody for 12 h at 4°C. For OPN staining, an additional incubation with a fluorophore conjugated secondary antibody was carried out in 0.05% (vol/vol) Triton X-100 PBS for 12 h at 4°C. Samples were then washed in 0.05% (vol/vol) Triton X-100 PBS at room temperature for 4 h. DAPI (0.1 μg/ml) was add in PBS for 4 h. Samples were subjected to a second round of fixation before performing washes in gradients (25%, 50%, 65%, 85%, 90%, 95%, and 100%) of methanol for 1 h each at room temperature followed by a wash in a series (50%, 75%, 100%) of benzoic acid:benzyl benzoate (Sigma-Aldrich) in a 1:2 ratio before being imaged in 3D on a LSM 880 (Zeiss) using Fast Airy scan mode.

Tert-butanol and benzoic acid:benzyl benzoate (Sigma Aldrich) at a 1:2 ratio were titrated to a pH of 9.5 with triethylamine. Tissue samples were placed in glassware with the prepared BABB solution for clearing for 7 hours at room temperature (Table 1). Cleared tissue can be stably stored in this solution at 4 °C or whole-mounted for immediate imaging.