Fig. 1. Effect of autofluorescence elimination reagents—AR and NeuN antibody penetration in processed human slices.

a, b Comparison of seven different autofluorescence elimination reagents and AR in 500 μm-SWITCH processed slices using LSFM with the excitation light at 405, 488, 561, and 638 nm. For H₂O₂ and CuSO₄, we examined three and two different concentrations, respectively. Statistical analysis (n = 20) was performed between two transformation protocol methods (SWITCH vs SHIELD) (a) and classic SWITCH-processed tissue vs autofluorescence elimination treatments on SWITCH-treated slices (a, b). A Mann–Whitney test was performed (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). Error bars show mean ± SD. Abbreviation: AR antigen retrieval, SB Sudan black. c, d Effect of temperature (4 °C, 37 °C) and tissue processing (SWITCH, SHIELD, and SHORT), e, f autofluorescence treatments (SB, NaBH₄, and CuSO₄) on NeuN immunofluorescence labeling. The plot profiles in c–f show the mean intensity ± SD of three different regions.