Fig. 5: AmpFISH assay in combination with IF.

a IF and AmpFISH assays were verified mutually. IF for CaMKIIα protein and AmpFISH for CaMKIIα mRNA were double-labeled in the same slice from the CPu brain region (scale bar: 20 μm). b The TH + CaMKIIα⁺ cell subtypes were detected in the slice from the VTA brain region through a combination of IF with AmpFISH (scale bar: 20 μm). c The proportion of positive double-labeled cells was analyzed. “Overlap” represents cells with both IF-and AmpFISH-positive signals. Overlap/FISH represents the proportion of overlapping cells with a positive AmpFISH signal (91.68 ± 2.07%, n = 5). In the same way, Overlap/FISH represents the proportion of overlapping cells with a positive IF signal (98.50 ± 1.14%, n = 5). The total number of “overlap” cells, cells with AmpFISH signal, and cells with IF signal were 170, 185, and 172, respectively, from five different brain slices. d Statistical analysis and comparison of cell populations in the SNC and VTA brain slices. The proportion of Th⁺/CaMKIIα⁺ cells (“overlap”) accounting for T⁺ cells in the VTA area (25.27 ± 0.46%) is significantly higher than that in the SNC area (13.97 ± 1.04%) (n = 4) (unpaired t-test with Welch’s correction, two-tailed, P < 0.001).