Extended Data Fig. 6: Validation of dye-ligand formulation and JFX₆₇₃-HTL, a bioavailable red dye-ligand with improved photostability.

a, Comparison of JF₆₆₉-HTL and JFX₆₇₃-HTL. Note the addition of deuterium and additional carbon in the R position of JFX₆₇₃. b, Bleaching curves of JF dyes showing normalized fluorescence over 30 bleaching cycles. Plotting mean and SD for n = 3 for each dye-ligand / timepoint c, JFX₆₇₃-HTL was significantly more photostable than the other dyes tested. Plotting mean and SD for n = 3 for each dye-ligand d, Example sections of MeCP2-HT mice do not show notable differences in brain availability of JF₆₆₉-HTL versus JFX₆₇₃-HTL. All panels show the pulse dye. e, Solubility of JF-HTL dyes using DMSO (20 μl), Pluronic F127 (20 μl), and PBS (60 μl) formulation over time after resuspension. No significant decrease in solubility was observed and all in vivo dyes used reached the intended solubility of 1 mM (2-way ANOVA [Dye-ligand x Time] p < 0.05; Time: F(3,20) = 0.66, p = 0.5835; Dye: F(4,20) = 149.21, p < 0.0001; Interaction: F(12,20) = 1, p = 0.4794). Plotting mean and SD for n = 3 for each dye-ligand / formulation. f, Example coronal slices from MeCP2-HaloTag animals injected with different ratios of DMSO and Pluronic F127. The original 1:1 ratio was the best, as shown by the brighter pulse dye-ligand staining. g, Different formulation than (e) to test the replacement of DMSO (left) with Captisol (middle) or a combination of Captisol and Pluronic F127 (right). We saw mixed effects on solubility, while some dyes (JFX₆₇₃-HTL) retained solubility, and some did not (JF₆₆₉-HTL; 2-way ANOVA, interaction: F(6,24) = 13.31, p 1.61e-6; Dye: F(3,24) = 67.35., p < 0.0001; Formulation: F(2,24) = 46.91, p = 1e-9). Plotting mean and SD for n = 3 for each dye-ligand / timepoint h, Example coronal slices from MeCP2-HaloTag animals injected with solutions from (f). Both dyes that were less soluble with Captisol alone or Captisol with Pluronic F127 (JF₆₆₉ left) or those that were as soluble (JFX₆₇₃ right) were less bioavailable without DMSO. n = 1 for each condition. i, Immunohistochemistry against GFAP (left) and Iba1(right) for 4 conditions, i-Positive control: A cortical lesioned animal. ii-Negative control: Naive animal without any manipulation. iii-Virus injection: An animal was injected with the GFP-HaloTag virus and perfused 3 weeks after injection. iv-Dye-ligand injection: An animal was injected with JF₆₆₉-HTL and perfused 24 h after injection. Both dye-ligand injection and virus injection do not increase GFAP or Iba1 levels. n = 1 for each condition.