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HALO® Module

ISH-IHC Quantification

(Designed for RNASCOPE®)

Indica Labs ISH-IHC measures one IHC stain and up to two chromogenic and/or silver-labeled DNA or RNA ISH probes on a cell-by-cell basis. This allows the user to rapidly contextualize the corresponding protein and gene expression profile of every cell across the tissue. The HALO ISH-IHC module is designed to work with up to 5 stains, including a nuclear stain and up to 4 IHC biomarkers or ISH probes and supports the H-score protocol for RNAscope as recommended by the manufacturer, ACD, a Bio-techne brand.

The module reports numerous outputs including, but not limited to: number of IHC positive cells in total and by cell compartment, Average IHC OD and average IHC OD by cell compartment, percentage IHC positive total and by cell compartment, number and percentage of probe 1 and/or probe 2 positive cells, probe 1 and probe 2 copies per cell and area per cell, cell score for probe 1 and probe 2 (0, +1, +2, +3 and +4), total H-score for each probe, total cells and percentage of total cells positive for IHC marker and probe 1 and/or probe 2 (dual and triple positive cells).   Histograms are automatically generated representing the frequency distribution of probes per cell [Figure 1] and RNAscope cell scoring [Figure 2] as shown below.

Figure 1 shows a distribution curve of probe number on a cell by cell basis, in this example most cells were shown to have between 0-5 copies per cell.

Figure 2. This bar chart shows the numbers of cells that were categorized as 0, 1+, 2+, 3+ and 4+ respectively. The chart shows that a slim majority of cells were positive for RNA probe expression.

The ISH-IHC quantification module integrates seamlessly into the HALO® platform which is compatible with a number of third party systems and  file formats.

File formats supported by the HALO image analysis platform:

  • Non-proprietary (JPG, TIF, OME.TIFF)
  • Nikon (ND2)
  • 3D Histech (MRXS)
  • Akoya (QPTIFF, component TIFF)
  • Olympus / Evident (VSI)
  • Hamamatsu (NDPI, NDPIS)
  • Aperio (SVS, AFI)
  • Zeiss (CZI)
  • Leica (SCN, LIF)
  • Ventana (BIF)
  • Philips (iSyntax, i2Syntax)
  • DICOM (DCM*)
    *whole-slide images

Quantitative RNAscope Image Analysis Guide​

Advance your RNAscope image analysis by downloading the latest Quantitative RNAscope Image Analysis Guide from Indica Labs and ACD, a Bio-techne brand.

RNAscope® Publications with the HALO® Image Analysis Platform

Read this publications list to see examples of research in areas from neuroscience to oncology using HALO to analyze RNAscope images.

Publication Spotlight

The table below includes publications that cite the ISH-IHC and FISH-IF modules. 

Your publication not on the list?  Drop us an email to let us know about it!

Title Authors Year Journal Application HALO Modules Product
Detection of EpsteinñBarr Virus in Periodontitis: A Review of Methodological Approaches Tonoyan L, Chevalier M, Vincent-Bungnas S, Marsault R, Doglio A 2021 Mircroorganisms Review ISH/FISH, ISH-IHC/FISH-IF HALO
Detection of engineered T cells in FFPE tissue by multiplex in situ hybridization and immunohistochemistry Wright JH, Huang L-Y, Weaver S, Archila LD, McAfee MS, Hirayama AV, Chapuis AG, Bleakley M, Rongvaux A, Turtle CJ, Chanthaphavong S, Campbell JS, Pierce RH 2020 Journal of Immunological Methods Immunology ISH/FISH, ISH-IHC/FISH-IF HALO
Epicardium-derived cells organize through tight junctions to replenish cardiac muscle in salamanders Eroglu E, Yen C, Witman N, Elewa A, Araus A, Wang H, Szattler T, Umeano C, Sohlmer J, Goedel A, Simon A, Chien K 2022 Nature Cell Biology Myology ISH/FISH, ISH-IHC/FISH-IF HALO
Satellite repeat RNA expression in epithelial ovarian cancer associates with a tumor immunosuppressive phenotype Porter R, Sun S, Flores M, Berzolla E, You E, Phillips I, KC N, Desai N, Tai E, Szabolcs A, Lang E, Pankaj A, Raabe M, Thapar V, Xu K, Nieman L, Rabe D, Kolin D, Stover E, Pepin D, Stott S, Deshpande V, Liu J, Solovyov A, Matulonis U, Greenbaum B, Ting D 2022 The Journal of Clinical Investigation Immuno-oncology ISH/FISH, ISH-IHC/FISH-IF HALO
Systemic Nos2 Depletion and Cox inhibition limits TNBC disease progression and alters lymphoid cell spatial orientation and density Somasundaram V, Ridnour L, Cheng R, Walke A, Kedei N, Bhattacharyya D, Wink A, Edmondson E, Butcher D, Warner A, Dorsey T, Scheiblin D, Heinz W, Bryant R, Kinders R, Lipkowitz S, Wong S, Pore M, Hewitt S, McVicar D, Anderson S, Chang J, Glynn S, Ambs S, Lockett S, Wink D 2022 Redox Biology Oncology Classifer, ISH/FISH, Spatial Analysis, Registration, ISH-IHC/FISH-IF HALO
Annexin A2/TLR2/MYD88 pathway induces arginase 1 expression in tumor-associated neutrophils Zhang H, Zhu X, Friesen T, Kwak J, Pisarenko T, Mekvanich S, Velasco M, Randolph T, Kargl J, Houghton A 2022 Journal of Clinical Investigation Immuno-oncology Spatial Analysis, ISH-IHC/FISH-IF HALO
Spatiotemporal transcriptome analysis reveals critical roles for mechano-sensing genes at the border zone in remodeling after myocardial infarction Yamada S, Ko T, Hatsuse S, Nomura S, Zhang B, Dai Z, Inoue S, Kubota M, Sawami K, Yamada T, Sassa T, Katagiri M, Fujita K, Katoh M, Ito M, Harada M, Toko H, Takeda N, Morita H, Aburatani H, Komuro I 2022 Nature Cardiovascular Research Myology ISH-IHC/FISH-IF HALO
Loss of functional System x-c uncouples aberrant postnatal neurogenesis from epileptogenesis in the hippocampus of Kcna1-KO mice Aloi M, Thompson S, Quartapella N, Noebels J 2022 Cell Reports Neuroscience ISH-IHC/FISH-IF HALO
Multiparameter immunohistochemistry analysis of HIV DNA, RNA and immune checkpoints in lymph node tissue Richardson Z, Deleage C, Tutuka C, Walkiewica M, Del Rio-Estrada P, Pascoe R, Evans V, Reyesteran G, Gonzales M, Roberts-Thomson S, Gonzalez-Navarro M, Torres-Ruiz, F, Estes J, Lewin S, Cameron P 2021 Journal of Immunological Methods Infectious Disease ISH-IHC/FISH-IF HALO
The potassium channel auxiliary subunit Kv_2 (Kcnab2) regulates Kv1 channels and dopamine neuron firing Yee J, Rastani A, Soden M 2022 Journal of Neurophysiology Neuroscience ISH-IHC/FISH-IF HALO
Transcriptional vulnerabilities of striatal neurons in human and rodent models of Huntington’s disease Matsushima A, Pineda S, Crittenden J, Lee H, Galani K, Mantero J, Tombaugh G, Kellis M, Heiman M, Graybiel A 2023 Nature Communications Neuroscience Area Quantification, ISH-IHC/FISH-IF HALO
Exploration of spatial heterogeneity of tumor microenvironment in nasopharyngeal carcinoma via transcriptional digital spatial profiling Wang L, Wang D, Zeng X, Zhang Q, Wu H, Liu J, Wang Y, Liu G, Poan Y 2023 International Journal of Biological Sciences Oncology Area Quantification, ISH-IHC/FISH-IF HALO

Related HALO Modules

TMA (Tissue Microarray) Add-On

Module to assist in the segmentation of TMA spots for further analysis.

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Tissue Classifier Add-On

Separate multiple tissue classes across a tissue using a learn-by-example approach. Can be used in conjunction with all other modules (fluorescent and brightfield) to select specific tissue classes for further analysis.

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Multiplex FISH (RNAscope®)

Quantify number, intensity and co-expression of an unlimited number of fluorescently labeled RNA or DNA probes on a per cell basis. Includes support for fluorescent RNAscope® assays.

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