Table 2.

Troubleshooting Table.

Step	Problem	Possible Reason	Solution
2	Lymphangiography injection	Difficulty in controlling the injection volume and force.	Injecting small amounts of liquid in a controlled way is challenging. We recommend using the injection apparatus described in the in the Reagent Setup (Figure S1). After filling the entire apparatus with saline, a small air bubble can be introduced in the tip of the injection needle. Then the fluorescent dye can be drawn into the tubing, with the air bubble separating the fluorescent dye and the saline. The distance the air bubble travels and the cross sectional area of the tubing can be used to measure the volume of dye injected.
		Difficulty in filling the PLV	Subcutaneously inject into the center of the footpad. Injection at the periphery will cause inefficient uptake of FITC-dextran.
3	Cannot locate lymphatic vessels	Unfamiliarity of location of PLV	For beginners, inject Evans Blue dye at the footpad instead of fluorescent dye using the injection apparatus. Lymphatic vessels will be highlighted in blue.
	Trouble immobilizing the leg for the surgery	Dermal tape is not sticking	Apply a modest amount of ethanol to skin with fingertips. Spraying ethanol will cause tape to lose adhesiveness, leading to inefficient surgery.
11	Lymphatic vessels are not clearly visible	Removal of fat is inadequate	Fat tissue may block the fluorescent signal during imaging and distort the optical image. Remove as much fat as possible with Dumont #5 forceps and blunt dissection. (See Step 5)
		Tissue injury during surgery	Make sure that no blood or lymphatic vessels are injured, resulting in blood hemorrhage or damage along the lymphatic vessels.
		Incorrect positioning of leg in petri dish	Make sure the leg is placed against the bottom of the petri dish. This allows for the vessel to remain in focus along its length. (See Step 6).
12	Lymphatic vessel contrast is too faint or too intense.	Volume of injected FITC-dextran was inappropriate.	Use a piece of polyethylene tubing to connect 30G × ½ needle. With this simple device, the injection can be gentle and the small volume can be controlled easily (See Reagent Preparation. Fig. S1).
		Site of injection was inaccurate	Inject into the center of the footpad. Injection at the periphery will cause inefficient uptake of FITC-dextran. (See Step 2).
13	Lymphatic vessels not contracting	Lymphatic vessel damage during surgery.	Overambitious removal of fat or rough handling of the surgical preparation can cause damage to the lymphatic vessels, leading to leakage of FITC-dextran or failure of lymphatic vessels to contract. During surgery, care should be taken not to puncture the lymphatic vessels or move it from its normal position.
		Sub-optimal environment	Wait at least 15 min for mouse to adjust to imaging conditions. Make sure the animal temperature is maintained at 37°C.
		Leg taped too tightly impairing vasculature	Taping the leg too tightly to the petri dish bottom can impair flow. Loosen the tape, while still keeping the PLV flat along the petri dish bottom (See Step 7).