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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

RNAscope® In Situ Hybridization Assay Workflow

  • Step 1:


    Tissue sections or cells are fixed onto slides and pretreated with RNAscope® Pretreatment Kit to unmask target RNA and permeabilize cells.

  • Step 2:


    Designed with ~20 target-specific double Z probes, RNAscope® Probes hybridize to target RNA molecules.

  • Step 3:


    RNAscope® Detection Reagents amplify the hybridization signals via sequential hybridization of amplifiers and label probes.

  • Step 4:


    Each punctate dot signal represents a single test target RNA molecule and can be visualized with a microscope.

  • Step 5:


    Single-molecule signals can be quantified on a cell-by-cell basis by manual counting or automated image analysis using HALO Software.

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

RNAscope® Multiplex Fluorescent v2

RNAscope® Fluorescent Multiplex

Plexing offered for target detection

12 Plex capability enabled with RNAscope® 12Plex Ancillary Kit (Cat. No. 324140)*

4 Plex capability enabled with RNAscope® 4-Plex Ancillary Kit for Multiplex Fluorescent v2 (Cat. No. 323120)*

3 Plex capability

Compatible sample preparation

Fresh frozen, Fixed frozen

FFPE, fixed frozen (low expressors)** Cells and fresh frozen (if highly autofluorescent)

Fresh frozen, Cells, Fixed frozen (high expressors)

Additional items required


Opal dyes must be purchased from Akoya Biosciences.


Workflow length

9 hr (Recommended to split over 3 days)

14 hr (Recommended to split over 2 days)

6-8 hours

Fluorophores used/recommended

Alexa Fluor-488, Atto-550, Atto-647N, Alexa Fluor-750 fluorophores provided in the detection kit (Fluoro reagents)

Purchased from Akoya Biosciences:
Opal™ 520 Reagent Pack (Cat No: FP1487001KT)
Opal™ 570 Reagent Pack (Cat No: FP1488001KT)
Opal™ 690 Reagent Pack (Cat No: FP1497001KT)

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)

Alexa Fluor-488, Atto-550 and Atto-647N fluorophores provided in the detection kit (Amp 4)

Cleavable Fluorophore




ISH-IF compatibility


Compatible: please refer to Doc# 323100-TN

Not recommended

Recommended for

Low autofluorescing tissues

High autofluorescing tissues

Low autofluorescing tissues

Probes Designation

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

RNAscope® Probes: C1, C2, C3, C4

RNAscope® Probes: C1, C2, C3

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 Max 550 nm/Em Max 570 nm (Cy3)
Ex Max 648 nm/Em Max 667 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)

Ex Max 358 nm/Em Max 461 nm (DAPI)
Ex Max 494 nm/Em Max 517 nm (FITC)
Ex Max 550 nm/Em Max 570 nm (Cy3)
Ex Max 648 nm/Em Max 667 nm (Cy5)

Mounting medium

Prolong Gold

Prolong Gold

Prolong Gold

Cat No





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

RNAscope® Fluorescent Multiplex Reagent Kit employs Alexa Fluor-488, Atto-550 and Atto-647N fluorophores for up to 3-plex detection capability.

Area of research


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


Contact Us

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Advanced Cell Diagnostics

Our new headquarters office starting May 2016:

7707 Gateway Blvd.  
Newark, CA 94560
Toll Free: 1 (877) 576-3636
Phone: (510) 576-8800
Fax: (510) 576-8798



19 Barton Lane  
Abingdon Science Park
OX14 3NB
United Kingdom
Phone 2: +44 1235 529449
Fax: +44 1235 533420



Advanced Cell Diagnostics China

Building 15, No. 26 Xihuan South Road
Beijing Economic-Technological Development Area
Beijing, China, Zip code: 100176

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