Step-by-step preparation of mouse eye sections for routine histology, immunofluorescence, and RNA in situ hybridization multiplexing

It can be challenging to maintain tissue integrity using established histology protocols. Here, we describe a protocol composed of Hartman’s fixation, window technique, microwave-based tissue processing, optimized depigmentation, and antigen retrieval pretreatment. This is followed by the ViewRNA single-molecule fluorescence in situ hybridization and immunofluorescence techniques to optimize routine histological staining and molecular histology multiplexing assays. Our protocol is highly reproducible in any laboratory and may decrease animal usage and lab resource expenditure. For complete details on the use and execution of this protocol, please refer to Pang et al. (2021).