Table 2.

Mapping drug distribution in tumor microenvironment

Process	Average Time	Notes
Drug Injection to mouse tumor model	-	-Intravenous injection of fluorescent macromolecular drug
Fixing, embedding, and macrosectioning	1 h	-Fixing tumor in 2% paraformaldehyde (PFA) for 10 min -Embedding tumor in 2% agarose gel -Macro-sectioning at 400 μm thickness -Fixing macrosections in 2% PFA for 5 min
Immunofluorescence staining	18 h	-Incubating with 4 different fluorescent primary antibodies at 4°C
Washing and fixing	40 min	-Washing with PBS x3 times, and fixing in 2% PFA for 10 min
Tissue clearing	3 h	-Incubating sequentially for 1 h each in 20%, 50%, 80% D-fructose solutions (containing 0.3% (v/v) α-thioglycerol) at 25°C
Confocal imaging	1 h/macrolayer (6×5×0.4 mm3)	-Using 10X objective (X/Y/Z grid resolutions=1.82/1.82/7.5 μm) -Using 5 different excitation and emission filters -Using 40X objective for high-resolution 3D imaging of interest area
Image processing and analysis	1 day	-Using Fiji software and created macros for 3D image reconstruction and analysis