Fig. 2

TSA-PACT allows signal amplification and SNR optimization. A A sketch of the structure of zebrafish brain. The box indicates the optic tectum, the imaged area for (B) and (D). B and C Three-dimensional images (B) and fluorescence intensity of the signals in different depth (C) in optic tectum with HuC/D staining post-IF-PACT/TSA-PACT. For different clearing methods, the same staining and imaging condition were used for the same regions. Samples were all incubated in 1 μg/mL HuC/D antibody. Images were acquired by 5 μm interval with the same microscope parameters (0.1% wave length; 100 μm pinhole; 600 V detector gain). Fluorescence intensity of signals was calculated from each 50 μm-z projection. The level of significance was calculated by unpaired Student’s t-test. (Voxel size: 0.624 μm × 0.624 μm × 5 μm; mean ± S.E.M.; n = 3; *p < 0.05; **p < 0.01). D and E Three-dimensional images (D) and signal-to-noise ratio in different depth (E) in optic tectum with HuC/D staining post-IF-PACT/TSA-PACT. Samples were incubated in the HuC/D antibody of 0.1, 1, 2, 5 or 10 μg/mL. Images were captured by 5 μm interval within the 300 μm depth, using the optimized parameters of confocal microscope (100 μm pinhole; 600 V detector gain; 1 μg/mL IF-PACT, 4% wave length; 2 μg/mL IF-PACT, 4% wave length; 5 μg/mL IF-PACT, 3% wave length; 10 μg/mL IF-PACT, 2% wave length; 0.1 μg/mL TSA-PACT, 0.7% wave length; 1 μg/mL TSA-PACT, 0.1% wave length; 2 μg/mL TSA-PACT, 0.1% wave length; 5 μg/mL TSA-PACT, 0.1% wave length). Signal-to-noise ratio was calculated from each 50 μm-z projection. The level of significance was calculated by one-way ANOVA test, followed by Bonferroni post hoc test, shown in Additional file 4: Table S3. (Voxel size: 0.624 μm × 0.624 μm × 5 μm; mean ± S.E.M.; n = 3)