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. 2024 Aug 5;15:6648. doi: 10.1038/s41467-024-50411-z

Fig. 2. Multicolor immunofluorescence enabled by scFv probes and linear unmixing of confocal micrographs.

Fig. 2

a Representative confocal images (n = 3 experiments in each category) of different sections from the cerebellum labeled with: a calbindin-specific scFv probe conjugated with Alexa Fluor 488, a VGluT1-specific scFv probe conjugated with Alexa Fluor 532, a GFAP-specific scFv probe conjugated with 5-TAMRA, a Kv1.2-specific scFv probe conjugated with Alexa Fluor 594, and a parvalbumin-specific scFv probe conjugated with Alexa Fluor 647. The double dotted lines delineate the Purkinje cell layer. (see Supplementary Figs. 12 and 15 for larger fields of view). CB calbindin, VGluT1 vesicular glutamate transporter 1, GFAP glial fibrillary acidic protein, Kv1.2 potassium voltage-gated channel subfamily A member 2, PV parvalbumin, TAM 5-TAMRA. b Workflow of multicolor imaging enabled by scFv probes and linear unmixing (see the text). c Representative maximum intensity projection of the multicolor fluorescence image stack acquired by linear unmixing of confocal images (n = 3 experiments). The signal of each fluorescent dye was pseudo-colored for better visualization. d Enlarged boxed inset from (c). The arrow indicates a Bergmann fiber (GFAP-positive) adjacent to the main dendrite of a Purkinje cell. Arrowhead indicates sites where axons form a pinceau structure labeled by the Kv1.2-specific scFv probe.