We also performed other combinations of multicolor staining and imaging: 4-color staining with BOBO-1, rat monoclonal anti-Sst IgG2b/Fab-Cy3, goat polyclonal anti-ChAT IgG/Fab-A594 and mouse monoclonal anti-Tph2 IgG1/Fab-A647 complexes (Supplementary Fig. 8a and Supplementary Movie 4) and 3-color staining of an Alzheimer’s disease model mouse (AppNL-G-F knock-in mouse)41 with SYTOX-G, mouse monoclonal anti-β-amyloid IgG1 (6E10)/Fab-A594 and mouse monoclonal anti-α-smooth muscle actin (SMA) IgG2a/Fab-A647 complexes (Supplementary Fig. 8b). The gene discussed is CHAT; the disease is early-onset autosomal dominant Alzheimer disease.