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HCR™ RNA-FISH Protocols

Multiplexed, quantitative, high-resolution RNA imaging

HCR™ RNA-FISH probe sets, amplifiers, and buffers enable multiplexed, quantitative, high-resolution RNA fluorescence in situ hybridization (RNA-FISH) with automatic background suppression throughout the protocol (how HCR™ RNA-FISH works). 

HCR™ RNA-FISH: Protocol Overview

Detection Stage

Amplification Stage


The same 2-stage protocol is used independent of the number of target RNAs.


Simple Robust Protocols

HCR™ RNA-FISH protocols are simple, robust, and enzyme-free, requiring only 2 stages independent of the number of target RNAs.

Automatic Background Suppression

HCR™ RNA-FISH reagents provide automatic background suppression throughout the protocol, ensuring that even if probes or hairpins bind non-specifically in the sample they will not generate amplified background, dramatically enhancing performance and ease-of-use.

Straightforward Multiplexing

HCR™ enables straightforward multiplexing using 1-step quantitative signal amplification for all target RNAs simultaneously.

Deep Sample Penetration

HCR™ amplification hairpins are short predominantly double-stranded oligos that penetrate thick samples to execute an enzyme-free amplification cascade at the site of the target.

Image Any Target mRNA. In Any Organism. In Any Sample Type. 

HCR™ offers unmatched versatility (see gallery): FFPE tissue sections, fresh/fixed frozen tissue sections, thick brain slices, cells in solution or on a slide, whole-mounts, cleared samples, model organisms, non-model organisms, environmental samples, or multi-kingdom samples. 

Custom Probe Design

Free custom probe design is available for any target mRNA in any organism across the tree of life.

Normal Sleep Schedule

We favor protocols with an overnight probe incubation step to enable researchers to maintain a normal sleep schedule; durations can be customized as desired based on sample thickness.


HCR™ IF + HCR™ RNA-ISH protocols enable multiplexed, quantitative, high-resolution protein and RNA imaging with 1-step HCR™ signal amplification for all protein and RNA targets simultaneously. 


Quantitative High-Resolution RNA Imaging

HCR™ RNA-FISH enables quantification of RNA targets with subcellular and single-molecule resolution in an anatomical context using two complementary imaging modes (see subcellular/single-molecule comparison):

Subcellular Quantitative RNA Imaging

Subcellular RNA imaging enables analog RNA relative quantitation with subcellular resolution in the anatomical context of thick autofluorescent samples.


Single-Molecule Quantitative RNA Imaging

Single-molecule RNA imaging enables digital RNA absolute quantitation with single-molecule resolution in the anatomical context of thick autofluorescent samples.

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