Table 1.

Tracking tumor perfusion and penetration

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

  • –Intravenous injection of fluorescent anti-CD31 antibody

Fixing, embedding, and macrosectioning2 h
  • –Fixing tumor in 4% PFA for 1 hour

  • –Embedding tumor in 2% agarose gel

  • –Macrosectioning at 400-μm thickness

Tissue clearing3 h
  • –Incubating sequentially for 1 hour each in 20%, 50%, 80% d-fructose solutions [containing 0.3% (v/v) α-thioglycerol] at 25°C

Confocal imaging30 min/macrolayer (6 × 5 × 0.4 mm3)
  • –Using 10× objective (X/Y/Z grid resolutions = 1.82/1.82/7.5 μm)

  • –Using 2 different excitation and emission filters

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

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