RNAscope Fluorescent Multiplex Assays

Spatial analysis of gene expression is an essential tool for comprehensive studies of complex, highly heterogeneous tissues, such as the brain, tumors, and developing organs. Traditional RNA-ISH can be challenging due to low sensitivity, inconsistent detection accompanied with limited plexing. RNAscope Fluorescent Multiplex assays provide exceptional sensitivity, allowing single-molecule detection of RNA targets at the single cell level. The RNAscope Multiplex Fluorescent assays are ideal for simultaneous detection studies of any genes in nearly any tissue-type using fluorescent labels. We offer with various levels of plexing to:

  • FIND cells of interest by using cell specific markers
  • CONFIRM cells by simultaneous detection of additional targets
  • CHARACTERIZE cellular heterogeneity of complex tissues by mapping and identifying multiple cell subtypes simultaneously


“Adding a histological context to scRNAseq data is important for fully understanding the biology of distinct cell populations, and HiPlex RNAscope is a great way to do this. The ability to stain a single section of tissue with up to 12 mRNA probes makes HiPlex RNAscope a powerful tool for distinguishing cells types in situ. While the HiPlex RNAscope protocol is relatively straightforward to conduct, ACD Bio has always been there to help when I need sales or technical assistance. I would recommend HiPlex RNAscope to anyone looking to visualize distinct populations of cells from scRNAseq data in situ.”
            -Jocob Swanson, Hospital for special surgery, NY

Which RNAscope Multiplex kit is best for me - three kits are available

Use the table below to learn more about which RNA in situ hybridization product is right for your research needs.

RNAscope HiPlex v2

RNAscope Multiplex Fluorescent v2

Plexing Capability

12 Plex

4 Plex

Reagent Kit

324400 RNAscope HiPlex12 Reagents Kit (488, 550, 650, 750) v2
324410 RNAscope HiPlex12 Reagents Kit (488, 550, 650) v2

323100 RNAscope Multiplex Fluorescent Reagent Kit v2

Area of research

Neuroscience, Oncology, Immuno-Oncology, Immunology

Neuroscience, Oncology, Immuno-oncology (CD8, PD1, PDL1)

Tissue Compatibility

FFPE, Fresh Frozen, Fixed Frozen

FFPE, Fixed Frozen (low expressors)** Cells and Fresh Frozen (if highly autofluorescent)

Recommended for

High and low autofluorescing tissues

High autofluorescing tissues

Assay TAT

9 hours

14 hours

Cleavable Fluorophore



ISH-IF compatibility


Compatible: please refer to Doc# 323100-TN

Probes Designation

RNAscope HiPlex Probes: T1, T2, T3, T4, ……T12

RNAscope Probes: C1, C2, C3, C4

Additional Reagents required


Opal dyes must be purchased from Akoya Biosciences.

Recommended Fluorophores

Alexa Fluor-488, Dylight 550, Dylight 650, Alexa Fluor-750 (These Fluorophores are provided in the Reagent Kit)

Opal™ 520 Reagent Pack (Cat No: FP1487001KT)
Opal™ 570 Reagent Pack (Cat No: FP1488001KT)
Opal™ 690 Reagent Pack (Cat No: FP1497001KT)
4-plex: Opal™ 520 Reagent Pack (Cat No: FP1487001KT)
Opal™ 570 Reagent Pack (Cat No: FP1488001KT)
Opal™ 620 Reagent Pack (Cat No: FP1495001KT)
Opal™ 690 Reagent Pack (Cat No: FP1497001KT)

Recommended Filter sets on the Fluorescent microscope:

Ex Max 358 nm/Em Max 461 nm (DAPI)
Ex Max 494 nm/Em Max 517 nm (FITC)
Ex 554 nm/Em 576 nm (Cy3)
Ex 644 nm/Em 669 nm (Cy5)
Ex Max 753 nm/Em Max 777 nm (Cy7)

Ex Max 358 nm/Em Max 461 nm (DAPI)
Ex Max 494 nm/Em Max 525 nm (FITC)
Ex Max 550 nm/Em Max 570 nm (Cy3)
Ex Max 588nm/Em Max 616 nm (Tx Red)
Ex Max 676 nm/Em Max 694 nm (Cy5.5)

Find more information at




RNAscope Fluorescent Multiplex Reagent Kit designed using ACD's proprietary amplification strategy. The assay uses Alexa Fluor-488, Atto-550, Atto-647N and Alexa Fluor-750 for detecting up to 12 targets on the same tissue section.

TSA-based Fluorescent RNAscope detection assay for up to 4-plex capability using Opal fluorophores (sold separately). Ideal for FFPE tissues

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Advanced Cell Diagnostics

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