Simplified integration of RNA and protein labeling for multiplex fluorescence microscopy in neuroscience models

This presentation will describe both common and specialized multiplex methods for RNA in situ hybridization (ISH) and immunohistochemical labeling of proteins (IHC). We initially used neuronal cell models to optimize and demonstrate the conditions needed for combining IHC and ISH.  In addition, we used FFPE and cryopreserved tissue models to introduce a method to address the challenges of integrating these two techniques, including specific and highly sensitive RNA detection with sample processing innovations for rapidly setting high refractive index media to facilitate fluorescence imaging. By employing these optimized ISH and IHC methods in normal murine brain sections and utilizing the Invitrogen™ EVOS™ M5000 Imaging System we observed expected spatial patterns of neural-relevant proteins and transcripts, allowing for both quantitative and qualitative evaluation of mRNA and protein expression. These approaches enable researchers to gain comprehensive insights into cellular microenvironments, cell-cell interactions, and disease mechanisms and are applicable to higher-plex spatial proteomics and transcriptomics studies.

Learning Objectives:

• How can I export images from the EVOS M5000 System?

• How can I retain IHC labeling after performing ISH using the ViewRNA protocol?

• How can I prevent bubble formation when using the ProLong RapidSet Mountant?

•  Is it possible to image more than four targets on the EVOS M5000 System?