Multiplexed Iterative FISH Experimental Protocol SOP002.v4.8 v1

This document SOP002, Multiplexed Iterative RNA FISH Experimental Protocol, describes the process for in-situ fluorescence labeling of RNA transcripts in cells and tissues using a layered (encoding + readout) probe design. This protocol can be used for single molecule RNA FISH or for high-throughput identity barcoding (MERFISH). This method uses a cleavable disulfide (S-S) reporter molecule, attached to a readout oligo, to allow for iterative rounds of RNA FISH staining and imaging of the same sample with minimal disruption to sample integrity between rounds. To improve the signal to noise, samples are gel embedded, cleared and photobleached. Tissue pre-processing including encoding probe labeling, gel embedding, photobleaching and clearing are described in Part 1 in addition to the in-situ staining method using automated fluidics (Part 2).