Fig. 3

TSA-PACT allows long-term signal retention and multiple molecular interrogations. A and B Three-dimensional images (A) and normalized mean fluorescence in different depth (B) in optic tectum with HuC/D staining taken at 0 and 16 months post IF-PACT/TSA-PACT. IF-PACT samples were incubated in 5 μg/mL HuC/D antibody and acquired images with 1% wave length, 100 μm pinhole and 650 V detector gain. TSA-PACT samples were incubated in 2 μg/mL HuC/D antibody and acquired images with 0.2% wave length; 41 μm pinhole; 560 V detector gain. Samples were held in 85% glycerol at 4 °C. After 16 months, they were re-analyzed using the previous microscope parameter. The level of significance was calculated by unpaired Student’s t-test. (Voxel size: 0.312 μm × 0.312 μm × 2 μm; mean ± S.E.M.; n = 5; **p < 0.01; and ***p < 0.001). C Images of multiple-round staining with anti-HuC/D antibody (green; first round) and anti-pHH3 antibody (red; second round). D Three-dimensional view of the whole brain with HuC/D and pHH3 staining in juvenile zebrafish. (Voxel size: 1.25 μm × 1.25 μm × 5 μm; green, HuC/D; red, pHH3)