Antigen flow through the lymphatic chain can be monitored with infrared fluorescent dye-labeled antigen. (A) Keyhole limpet hemocyanin (KLH) was labeled with 800CW and run on a polyacrylamide gel. (B) A linear correlation was found between the amount of KLH-800 and the fluorescent signal intensity (Fluorescent antigen signal is expressed as “arbitrary units (A.U.)). (C, D) Increasing amounts of KLH-800 were injected subcutaneously into the dorsum of the hind paw of a Balb/c mouse. The animal was imaged after 1 h; increasing amounts of KLH-800 correlated with an increasing fluorescent signal located at both the (C) popliteal and (D) sciatic lymph nodes. Data represent mean ± SEM, n = 5 per concentration. A similar increase in signal was observed in (E) popliteal and (F) sciatic lymph nodes extracted from mice injected with increasing amounts of KLH-800 and minced into T-Per. Data represent mean ± SEM, n = 5 per concentration. (G) A linear correlation was found between in situ signal and in-plate signal with an R-squared value of 0.7634. The dotted line indicates the 95% prediction band.