Extended Data Fig. 4: Calibration and validation of GFP-HT based JF dye-ligand screening in vivo.

a, Purified HT was added at saturation to a HTL JF (left: JF₆₆₉-HTL, middle: JF₅₈₅-HTL, right: JF₅₅₂-HTL) in an 8-well coverslip with 20 μL/well at the following concentrations (μM): 10, 5.0, 2.5, 1.25, 0.625, 0.3125, 0.15625, 0. All 8 wells were imaged under the same conditions as the fixed tissue slides (far red channel for JF₆₆₉ and red channel for JF₅₅₂ and JF₅₈₅). The offset at zero was subtracted and is reported in each panel. A linear slope (red line) was fitted to the data (blue circles) without an intercept term (JF₆₆₉-HTL: r² = 0.998; JF₅₈₅-HTL: r² = 0.9982; JF₅₅₂-HTL r² = 0.9963). This calibration covers 20 out of the 30 animals used and a naive calibration was used for the rest of the dyes (150 offset, 2000 slope). b, Left, mean Fraction Pulse is not correlated with the sum of the virus signal (GFP channel) per animal (Magenta is JF₆₆₉-HTL, red is JF₅₅₂-HTL, black is other dyes; r² = 0.02, two-sided Pearson correlation test p = 0.45, n = 28). An animal that was not injected with a virus (black square) has at least an order of magnitude less summed fluorescence. Right, mean Fraction Pulse is not correlated with the number of cells detected per animal (r² = 0.005, two-sided Pearson correlation test p = 0.717, n = 28; normalized by the area of tissue imaged). An animal that was not injected with a virus (black square) has at least an order of magnitude less detected cells. c, As the Fraction Pulse increases, so is the ratio between the Pulse and GFP, indicating that variability in the Chase cannot explain the increases in Fraction Pulse (Left: JF₅₅₂-HTL: n = 7, r² = 0.945, two-sided Pearson correlation test F(5) = 85.5, p = 0.00025; Right: JF₆₆₉-HTL: n = 10, r² = 0.65, two-sided Pearson correlation test F(8) = 14.7, p = 0.00495). X-axis: Fraction Pulse [Pulse / (Chase + Pulse)]. This compares the pulse injected in vivo to the total protein calculated by using the two dyes with their calibration. Y-axis: Pulse to GFP ratio. This compares the pulse injected in vivo to the total protein as estimated by the GFP fluorescence. The fact that these 2 ways correlate, means that we are obtaining reliable estimates for the dye-ligand calibrations (needed to calculate Pulse + Chase).