Table 1.

Overview of T3 procedure for core needle biopsy and average times required for each step

PROCESS	AVERAGE TIME	NOTES
Harvesting core needle biopsy and fixing	10 min	-Using a core biopsy needle (e.g. 18 gauge) -Fixing in 2% PFA for 5 min at 25°C -Washing in PBS
Immunofluorescence staining	18 h	-Incubating with fluorescent primary antibodies at 4°C -Washing in PBS -Fixing in 2% PFA for 5 min at 25°C
Optical clearing	2 h	-Incubating sequentially with 20% (30 min), 50% (30 min), 100% (1 h) D-fructose solutions (containing 0.3% (v/v) α-thioglycerol) at 25°C
Confocal microscopic imaging	1 h 30 min/biopsy (10X objective, 18 gauge biopsy of 0.5 cm length and 0.84 mm diameter)	-Using a 10X objective and voxel dimensions of X/Y/Z = 1.82/1.82/7.5 μm -Using 6 excitation and emission filters -Using a 40X objective for high-resolution 3D imaging of area of interest
Image processing and analysis	1 day	-Using commercial and open-source software, and custom macros for image reconstruction, deconvolution, and 3D analysis
Reverting biopsy	2 h	-Incubating sequentially with 50% (30 min), 20% (30 min), 0% (1 h) D-fructose solutions at 25°C
FFPE and IHC	~3–5 days	-Dehydration, paraffin embedding, sectioning (5 μm), IHC staining, light microscopy