Spatial analysis of organ-wide RNA, protein expression, and lineage tracing in the female mouse reproductive tract

Visualizing precise spatial patterns of an organ-wide gene and protein expression among diverse cell types can provide critical insights into the fundamental processes underlying normal tissue homeostasis and disease development. Here, we describe an optimized protocol for single-molecule RNA in situ hybridization (smRNA-ISH), immunohistochemistry, and cell lineage analysis of the female reproductive tract organs using commercially available smRNA-ISH probes, antibodies, and inducible Cre-mice. The high-resolution multispectral fluorescence imaging is performed using wide-field epifluorescence or confocal microscopy combined with a slide scanner. For complete details on the use and execution of this protocol, please refer to Chumduri et al. (2021). [Display omitted] •Tissue preparation for fixation of female reproductive tract (FRT) organ•The optimized protocol for labeling of the protein and RNA in the FRT tissue•Spatial analysis of gene, protein expression, and lineage development in FRT Visualizing precise spatial patterns of an organ-wide gene and protein expression among diverse cell types can provide critical insights into the fundamental processes underlying normal tissue homeostasis and disease development. Here, we describe an optimized protocol for single-molecule RNA in situ hybridization (smRNA-ISH), immunohistochemistry, and cell lineage analysis of the female reproductive tract organs using commercially available smRNA-ISH probes, antibodies, and inducible Cre-mice. The high-resolution multispectral fluorescence imaging is performed using wide-field epifluorescence or confocal microscopy combined with a slide scanner.