Protecting RNA Integrity for Spatial Transcriptomics While Improving Immunofluorescent Staining Quality

In comparison to bulk sequencing or single cell sequencing, spatial transcriptomics preserves the spatial information in tissue slices and can even be mapped to immunofluorescent stainings. This enables to unravel complex interactions of neighboring cells or to link cell morphology to transcriptome data. The 10x genomics visium spatial assay offers to combine spatial transcriptomics with immunofluorescent staining of cryo-sectioned tissue slices. We applied this technique to 10 µm fresh frozen mouse brain slices and developed a protocol that still protects RNA integrity while improving buffers for immunofluorescent staining. We investigated the influence of various parameters on RNA integrity and on antibody binding of blocking, staining as well as wash buffers. Finally, we propose a protocol that does not alter RNA integrity in comparison the 10x genomics demonstrated protocol but allows the usage of several antibodies that have not been compatible with the protocol before. In addition, we discuss the influence of the tested parameters which offers further development of application specific staining protocols. Transcriptome data of a newly established immunostaining protocol improving spatial transcriptomic applications Treatment PCI stands for Peritoneal contamination and infection.