Table 1 Overview of T3 procedure and average times required for each step.
From: Multiplex three-dimensional optical mapping of tumor immune microenvironment
Process | Average Time | Notes |
|---|---|---|
Fixing, embedding, and macrosectioning | 1 h | -Fixing tumor in 2% paraformaldehyde for 10 min |
-Embedding tumor in 2% agarose gel | ||
-Macrosectioning at 400 μm thickness | ||
-Fixing macrosections in 2% paraformaldehyde for 5 min | ||
Immunofluorescence staining | 18 h | -Incubating macrosections with 5 different fluorescent primary antibodies at 4 °C |
Washing and fixing | 40 min | -Washing macrosections with PBS × 3 times, and fixing in 2% paraformaldehyde for 10 min |
Tissue clearing | 3 h | -Incubating macrosections 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/section | -Mounting macrosections between coverslips |
-Scanning full volume (6 × 5 × 0.4 mm3) with 10X objective (X/Y/Z grid resolutions = 1.78/1.78/6.25 μm), using 5 different excitation and emission settings | ||
Image processing and analysis | 1 day | -Using Fiji software and created macros for 3D image reconstruction and analysis |
