Table 2.

Mapping drug distribution in tumor microenvironment

ProcessAverage timeNotes
Drug injection to mouse tumor model
  • –Intravenous injection of fluorescent macromolecular drug

Fixing, embedding, and macrosectioning1 h
  • –Fixing tumor in 2% PFA for 10 min

  • –Embedding tumor in 2% agarose gel

  • –Macrosectioning at 400-μm thickness

  • –Fixing macrosections in 2% PFA for 5 min

Immunofluorescence staining18 h
  • –Incubating with 4 different fluorescent primary antibodies at 4°C

Washing and fixing40 min
  • –Washing with PBS 3 times, and fixing in 2% PFA for 10 min

Tissue clearing3 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 imaging1 h/macrolayer (6 × 5 × 0.4 mm3)
  • –Using 10× objective (X/Y/Z grid resolutions = 1.82/1.82/7.5 μm)

  • –Using 5 different excitation and emission filters

  • –Using 40× objective for high-resolution 3D imaging of interest area

Image processing and analysis1 day
  • –Using Fiji software and created macros for 3D image reconstruction and analysis