Strong Opal Fluorescence Signal Detection During RNAscope

Cell & Tissue Engineering, Imec
Research Scientist

Overall

Quality of Results

Ease-of-Optimization

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Company:

Akoya Biosciences

Product Name:

Opal 570 Reagent

Catalog Number:

FP1488001KT

Clone Number:

Opal 570 dye was used to detect p21 or p16 mRNA during RNA in situ hybridization (RNAscope) in rat brain tissue and peripheral blood mononuclear cells (PBMCs).

Experimental Design and Results Summary

Applications

RNA in situ hybridization (RNAscope)

Sample

rat brain tissue (FFPE), heart and peripheral blood mononuclear cells (PBMCs)

Primary Incubation

AMP hibridization probes from RNAscope kit

Blocking Agent

none

Secondary Incubation

Opal 570 reagent (1:1000) in TSA Buffer, 30 min at 40 °C

Tertiary Incubation

none

Detection

Confocal

Results Summary

Recommended dilutions range from 1:750 to 1:3000, although fluorophore dilution may require optimization depending on the tissue type and experimental conditions. The image shows red fluorescent puncta in rat heart tissue corresponding to cdkn1a mRNA (the gene encoding p21). Images were acquired using a ZEISS LSM 710 confocal microscope equipped with a 40× objective lens.

DOI or PMID #

https://doi.org/10.1016/j.jconrel.2026.115174; 10.1093/cvr/cvaf208

Additional Notes

For PBMCs, there is a specific protocol (RNAscope® Fluorescent Assay for PBMC and Non-Adherent Cells).

Image Gallery

Summary

The Good

Great detection using a high dilution.

The Bad

none

The Bottom Line

Expensive, but it works very well at lower volumes than the ones recommended by the manufacturer.

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