biostudies-literatureChoi ELNIDDK NIH HHSNational Institute of Diabetes and Digestive and Kidney DiseasesMayo ClinicNational Institutes of Health103828https://www.ebi.ac.uk/biostudies/studies/S-EPMC12143785biostudies-literatureUnknown6(2)Gastrointestinal motor functions are orchestrated by enteric neurons that display a high degree of anatomical and functional variability. Here, we present a protocol for AI-supported immunofluorescence colocalization analysis in human enteric neurons. We describe steps for human gastric tunica muscularis preparation, immunofluorescent labeling of enteric neurons, and quantification of marker colocalization using Nikon NIS-Elements. This protocol addresses the challenges presented by the high density of neurons within enteric ganglia, overlapping fluorescence signals, background fluorescence, and potential user bias in quantification. For complete details on the use and execution of this protocol, please refer to Gao et al.¹.STAR protocolsProtocol for AI-supported immunofluorescence colocalization analysis in human enteric neurons.PMC12143785R01 DK131455R01 DK126827P01 DK068055R01 DK121766P30 DK084567Quiroz ASHayashi YOrdog TZhang YBharucha AEHuynh NMChoi ELGao FfalseProtocol for AI-supported immunofluorescence colocalization analysis in human enteric neurons.Gastrointestinal motor functions are orchestrated by enteric neurons that display a high degree of anatomical and functional variability. Here, we present a protocol for AI-supported immunofluorescence colocalization analysis in human enteric neurons. We describe steps for human gastric tunica muscularis preparation, immunofluorescent labeling of enteric neurons, and quantification of marker colocalization using Nikon NIS-Elements. This protocol addresses the challenges presented by the high density of neurons within enteric ganglia, overlapping fluorescence signals, background fluorescence, and potential user bias in quantification. For complete details on the use and execution of this protocol, please refer to Gao et al.¹.2025-01-01T00:00:00Z2025 Jun2026-07-02T03:20:15.569Z2026-07-02T03:12:18.733ZS-EPMC121437854038277310.1016/j.xpro.2025.103828