- Microinjection buffer: 48 mM K 2 HPO 4 , 4.5 mM KH 2 PO 4 ,
and 14 mM NaH 2 PO 4 , pH 7.2 (seeNote 2). - Glass bottom dish (e.g., MatTek).
- Microcentrifuge.
2.5 Microscope and
Imaging Software
- An inverted widefield fluorescence microscope. We use an
Olympus IX83 Motorized inverted fluorescence microscope
equipped with a back-illuminated EMCCD camera (Andor)
and an MT-20E excitation source (Olympus). - 400.95NA objective lens.
- Filter set for DAPI, EGFP, and TAMRA (e.g., Olympus
MT20). - Filter set for Cy5 (ET620/60, ET700/75m, and T660lpxr).
- Filter set for IRDye®800 (ET710/75x, ET810/90m, and
T760lpxr). - cellSens Dimension image acquisition software (Olympus).
- Fiji software for image analysis (seeNote 3).
2.6 Synthesis of
Reference-Dye
Labeled Dextran
- IRDye®800-NHS ester (e.g., Li-Cor) dissolved in anhydrous
DMSO or DMF (seeNote 4). - Reaction buffer: 50 mM sodium borate buffer, pH 8.
- Elution buffer: 48 mM K 2 HPO 4 , 4.5 mM KH 2 PO 4 , and
14 mM NaH 2 PO 4 , pH 7.2. - Aminodextran solution: 2.5 mg/mL aminodextran
(MW¼10 kDa) in reaction buffer. - Gel chromatography columns (e.g., GE Healthcare NAP-5).
- Centrifugal filter units, YM-3, 3000 MW cutoff (e.g., Millipore
Centricon). - Microcentrifuge.
- Thermomixer.
- Spectrophotometer (e.g., Thermo Scientific BioMate 3S).
2.7 Preparation of
Microemulsion
Samples
- Span-80.
- Tween 80.
- Mineral oil.
- 5 mL glass vials.
- 50 mL tubes.
- Magnetic stirrer and magnetic stir bars.
- Glass pipettes.
- Electronic scale.
246 Mingming Chen et al.