Fig. 3: In situ detection of proteins and neurotransmitters by π-FISH rainbow.

a Schematic for in situ detection of multiple proteins and neurotransmitters by π-FISH rainbow. (i) Antibodies were conjugated to barcoded ssDNA and then incubated with target proteins and neurotransmitters. (ii) Hybridization of probes with barcoded ssDNA and signal amplification using π-FISH rainbow. b Detection of CSFV E2 protein in HeLa cells, transfected with the rAAV-E2 plasmid, by π-FISH rainbow and conventional immunostaining, respectively. Scale bar, 10 μm. c Intensity surface plot analysis of (b). Cells lacking E2 protein and background are indicated by oval region and triangular area, respectively. d Detection of pol II protein by conventional immunostaining (top) and π-FISH rainbow (bottom) with different dilutions of primary antibody (1:100, 1:1000, 1:5000, and 1:10,000), respectively. Scale bars, 10 μm. e Quantification of mean signal intensity per cell of (d). n  =  20 cells per group. Data were expressed as mean ± s.e.m. Two-tailed unpaired t test was used to compare two groups (1:100, P = 0.0137; 1:1000, P =  1.64E-29; 1:5000, P =  9.82E-38; 1: 10,000, P =  1.89E−39). *P < 0.05; ****P < 0.0001. Source data are provided as a Source Data file. f Simultaneous detection of CSFV E2, PCV2 Cap, IFNG-flag, and pol II proteins in CSFV and PCV2 co-infected PK-15 cells by π-FISH rainbow. Scale bars, 10 μm. g Codetection of GABA and NeuN in mouse cerebral cortex by π-FISH rainbow. Yellow arrow indicated cells co-expressing GABA and NeuN, and the white arrow indicated cells expressing only NeuN. The boundary of the cortex was indicated by white dashed lines. Scale bars, 150 μm.