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Rnascope 2.5 hd assay red kit

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The RNAscope 2.5 HD Assay-RED kit is a highly sensitive and specific in situ hybridization (ISH) technology designed for the detection and localization of RNA targets in fixed tissue samples. The kit utilizes a proprietary signal amplification system to enable the visualization of single RNA molecules within individual cells.

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Rnascope assay, by Advanced Cell Diagnostics (4 mentions) Bx60 microscope, by Nikon (2 mentions) Lsm 710, by Zeiss (2 mentions) Mach 4 mr ap, by Biocare Medical (2 mentions) Ferangi blue, by Biocare Medical (2 mentions) Integrator system, by Visiopharm (1 mentions) Tcs sp5 confocal microscope, by Leica (1 mentions) Novolink polymer detection system kit, by Leica (1 mentions) Xylene, by Bio-Optica (1 mentions) Cm3050, by Leica (1 mentions) Rodent block m, by Biocare Medical (1 mentions) Goat anti mouse nkp46, by R&D Systems (1 mentions) Rat anti mouse ly6g, by BD (1 mentions) Dab chromogen kit, by Biocare Medical (1 mentions)

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RNAscope kit (59 citations) RNAscope assay (49 citations) RNAscope 2.5 HD RED kit (27 citations) RNAscope 2.5 HD Assay – RED (24 citations) RNAScope 2.5 HD Assay (12 citations) RNAscope 2.5 Assay (11 citations) RNAscope RED 2.5 kit (7 citations) RNAscope 2.5 Red assay kit (5 citations) RNAscope 2.5 HD –RED), (3 citations) RNAscope 2.5 assay kit (3 citations)

19 protocols using rnascope 2.5 hd assay red kit

1

SARS-CoV-2 Spike Protein in Situ Hybridization

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RNA in situ hybridization was performed via RNAscope 2.5 HD Red Assay Kit (Advanced Cell Diagnostics, Cat: 322350) in accordance with manufacturer’s instructions. Fixed tissue sections were treated with the manufacturer’s Fresh Frozen Tissue Sample Preparation Protocol, fixed in chilled 4% PFA, dehydrated, and treated with H2O2 and Protease IV before probe hybridization. Paraffinized sections were deparaffinized and treated with H2O2 and Protease Plus prior to hybridization. Probes targeting SARS-CoV-2 spike (Cat: 848561), positive control Hs-PPIB (Cat: 313901), or negative control DapB (Cat: 310043) were hybridized followed by proprietary assay signal amplification and detection. Tissues were counterstained with Gill’s hematoxylin. An uninfected mouse was used as a negative control and stained in parallel. Tissues were visualized using an Olympus BX60 microscope and imaged with a Nikon (Model #) camera.
Olivarria G.M., Cheng Y., Furman S., Pachow C., Hohsfield L.A., Smith-Geater C., Miramontes R., Wu J., Burns M.S., Tsourmas K.I., Stocksdale J., Manlapaz C., Yong W.H., Teijaro J., Edwards R., Green K.N., Thompson L.M, & Lane T.E. (2021). Microglia do not restrict SARS-CoV-2 replication following infection of the central nervous system of K18-hACE2 transgenic mice. bioRxiv.
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2

RNA ISH for IFIT1 and CLEC4C in Kidney Tissue

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RNA in situ hybridization (ISH) for the IFN-induced gene IFIT1 and the pDC marker gene C-type lectin domain family 4 member C (CLEC4C, also known as BDCA2) was performed using RNAscope® 2.5 HD Red assay kit (Advanced Cell Diagnostics) and RNAscope probe specific to the gene encoding Homo sapiens (Hs) IFIT1 transcript variant 3 mRNA and Hs CLEC4C transcript variant 2 mRNA, according to the manufacturer’s instructions. Briefly, 5 μm formalin-fixed paraffin embedded biopsied kidney tissue sections were pretreated with heat and proteases prior to hybridization with the target oligo probes. Preamplifiers and amplifiers were sequentially hybridized to the target probe, and alkaline phosphatase-based amplifiers were then hybridized in final steps followed by detection using chromogenic substrate. Samples were quality-controlled for RNA integrity with an RNAscope probe specific to Hs-PPIB RNA and for background signal using bacterial negative control probe DapB. Specific RNA staining signal was identified as red, punctate dots. ISH for IFIT1 mRNA and CLEC4C mRNA was done in serial sections for every sample. Quantitative measurement of mRNA expression (i.e. IFIT1 and CLEC4C) and heatmap analysis was performed by Visiopharm Integrator System (Visiopharm).
Iwamoto T., Dorschner J.M., Selvaraj S., Mezzano V., Jensen M.A., Vsetecka D., Amin S., Makol A., Osborn T., Moder K., Chowdhary V.R., Izmirly P., Belmont H.M., Clancy R.M., Buyon J.P., Wu M., Loomis C.A, & Niewold T.B. (2021). High Systemic Type I Interferon Activity is Associated with Active Class III/IV Lupus Nephritis. The Journal of rheumatology, 49(4), 388-397.
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3

SARS-CoV-2 Spike Protein RNA In Situ Hybridization

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RNA in situ hybridization was performed via RNAscope 2.5 HD Red assay kit (Advanced Cell Diagnostics, Cat: 322350) in accordance with manufacturer’s instructions. Fixed tissue sections were treated with the manufacturer's Fresh Frozen Tissue Sample Preparation Protocol, fixed in chilled 4% PFA, dehydrated, and treated with H2O2 and Protease IV before probe hybridization. Paraffinized sections were deparaffinized and treated with H2O2 and Protease Plus prior to hybridization. Probes targeting SARS-CoV-2 spike (Cat: 848561), positive-control Hs-PPIB (Cat: 313901), or negative control DapB (Cat: 310043) were hybridized followed by proprietary assay signal amplification and detection. Tissues were counterstained with Gill’s hematoxylin. An uninfected mouse was used as a negative control and stained in parallel. Tissues were visualized using an Olympus BX60 microscope and imaged with a Nikon (Model number) camera.
Olivarria G.M., Cheng Y., Furman S., Pachow C., Hohsfield L.A., Smith-Geater C., Miramontes R., Wu J., Burns M.S., Tsourmas K.I., Stocksdale J., Manlapaz C., Yong W.H., Teijaro J., Edwards R., Green K.N., Thompson L.M, & Lane T.E. (2022). Microglia Do Not Restrict SARS-CoV-2 Replication following Infection of the Central Nervous System of K18-Human ACE2 Transgenic Mice. Journal of Virology, 96(4), e01969-21.
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4

Quantifying IL17RA Expression in Tissue

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Animals were transcardially perfused with cold PBS. Brains were extracted and embedded in optimal cutting temperature (OCT) compound on dry ice. Sections were cut at 20μm thickness on a cryostat. In situ hybridizations were performed using RNAscope 2.5 HD Assay-Red kit (322350, Advanced Cell Diagnostics) using a probe targeting the Il17ra transcript (Mm-Il17ra-O1, 566131, Advanced Cell Diagnostics). The probe was designed to target region 444–882 of the Il17ra transcript (NM_008359.2). Modifications to the kit protocol to improve adherence of tissue to the slide include an extension of fixation time to 30min and the addition of a humidified bake step at 40°C immediately prior to probe hybridization. Sections were counterstained with DAPI. Images were acquired using a confocal microscope (LSM710, Carl Zeiss) with a 10x or 20x objective lens. Il17ra and DAPI expression was quantified using QuPath30 (link). Cells were divided into the following categories based on level of Il17ra expression: low = 1–3 puncta, medium = >3–9 puncta, high = >9–15 puncta, highest = >15 puncta.
Reed M.D., Yim Y.S., Wimmer R.D., Kim H., Ryu C., Welch G.M., Andina M., King H.O., Waisman A., Halassa M.M., Huh J.R, & Choi G.B. (2019). IL-17a promotes sociability in mouse models for neurodevelopmental disorders. Nature, 577(7789), 249-253.
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5

In-situ Detection of SARS-CoV-2 Replication

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For the in-situ hybridization for SARS-CoV-2 on the FFPE materials of the lung specimens, two probes were used to detect infected cells and identify replicating viruses (positive/plus- and negative/minus-sense). Positive and negative controls were integrated to verify the correctness of the staining results (RNAscope Probe–V-nCoV2019-S and V-nCoV2019-S-sense, Positive Control Probe–Hs-UBC, Advanced Cell Diagnostics, Germany) RNAscope 2.5 HD Assay-RED kit (Advanced Cell Diagnostics, Germany) was used for realizing the hybridization of the probes and its visualization according to the manufacture’s protocols by manual technique on 5 μm thick FFPE sections.
RNA-ISH evaluation was performed on the same samples (lungs in all patients and for other organs, which were tested positive by RT-qPCR) as the immunohistochemical SARS-CoV-analysis. The classification scheme was also identical.
Hirschbühl K., Dintner S., Beer M., Wylezich C., Schlegel J., Delbridge C., Borcherding L., Lippert J., Schiele S., Müller G., Moiraki D., Spring O., Wittmann M., Kling E., Braun G., Kröncke T., Claus R., Märkl B, & Schaller T. (2021). Viral mapping in COVID-19 deceased in the Augsburg autopsy series of the first wave: A multiorgan and multimethodological approach. PLoS ONE, 16(7), e0254872.
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6

Dvl-2 RNA Expression Profiling

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RNA in situ hybridization (ISH) was carried out using the RNAscope 2.5 HD Assay-RED KIT (324510, Advanced Cell Diagnostics, CA) according to the manufacturer's instructions. Mouse Dvl-2 (Mm-Dvl-2-C1, 1038481-C1), negative and positive control probes were purchased from Advanced Cell Diagnostics. See Supplementary Materials.
Sheng M., Lin Y., Xu D., Tian Y., Zhan Y., Li C., Farmer D.G., Kupiec-Weglinski J.W, & Ke B. (2021). CD47-MEDIATED HEDGEHOG/SMO/GLI1 SIGNALING PROMOTES MESENCHYMAL STEM CELL IMMUNOMODULATION IN MOUSE LIVER INFLAMMATION. Hepatology (Baltimore, Md.), 74(3), 1560-1577.
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7

ASCC3 Isoform Expression by RNAscope

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RNASCOPE 2.5 HD Assay Red Kit and probes targeting the long and short isoform of ASCC3 were designed and supplied by Advanced Cell Diagnostics. Cells were grown on slides and either left untreated or UV-irradiated with 15 J/m2 followed by 20 hr incubation. Cells were washed in PBS, fixed in 10% Neutral Buffered Formalin for 30 min. Fixed cells were then washed with PBS followed by dehydration in ethanol according to manufacturers protocol. Following dehydration, slides were stored in 100% ethanol at −20 C. Probe hybridization was performed strictly according to manufacturers protocol and counterstained with DAPI. Images were acquired using a Leica TCS SP5 confocal microscope.
Williamson L., Saponaro M., Boeing S., East P., Mitter R., Kantidakis T., Kelly G.P., Lobley A., Walker J., Spencer-Dene B., Howell M., Stewart A, & Svejstrup J.Q. (2017). UV Irradiation Induces a Non-coding RNA that Functionally Opposes the Protein Encoded by the Same Gene. Cell, 168(5), 843-855.e13.
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8

In situ Hybridization and IHC Analysis

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In situ hybridization was performed using an RNAScope 2.5 HD Assay-RED kit (Advanced Cell Diagnostics, Newark, CA, USA) following the manufacturer’s protocol. Gene-specific probes for human LOXL1-AS1 with human peptidyl-prolyl cis-trans isomerase B (PPIB) as a positive control and bacterial dapB as a negative control were designed by the manufacturer. RNA expression was determined by automated detection of chromogenic dots using the Color Deconvolution and Weka Classifiers plug-ins and was normalized to the positive control RNA signal. IHC staining was performed with an anti-Mycn antibody (cat. no. #51705, Cell Signaling Technology) and Novolink Polymer Detection System kit (Leica Biosystems, Richmond, IL, USA) according to the manufacturer’s instructions. Protein expression was determined by quantification of the chromogenic signal intensity. All images were analyzed with Fiji/ImageJ software.
Do A.D., Wu K.S., Chu S.S., Giang L.H., Lin Y.L., Chang C.C., Wong T.T., Hsieh C.L, & Sung S.Y. (2024). LOXL1-AS1 contributes to metastasis in sonic-hedgehog medulloblastoma by promoting cancer stem-like phenotypes. Journal of Experimental & Clinical Cancer Research : CR, 43, 130.
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9

RNAscope ISH Quantification of MNV-1 in Intestine

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RNAscope ISH assays were performed using the RNAscope 2.5 HD Assay-RED kit according to manufacturer’s instructions (Advanced Cell Diagnostics, Newark, CA). Formalin-fixed, paraffin-embedded serial sections of intestinal swiss rolls from mock- and MNV-1-infected pups were deparaffinized by heating at 60 °C for 30 min followed by xylene treatment and dehydration. Sections were hybridized with custom-designed probes targeting positive-sense or negative-sense MNV-1 RNA for 2 h at 40 °C prior to probe amplification and detection8 (link). Sections were counterstained with 50% hematoxylin to visualize tissue morphology and imaged using a Nikon DS-Fi2 color camera and NIS Elements software at the University of Florida Cell and Tissue Analysis Core. Sections were scored based on amount of virus present in the GALT, intestinal lamina propria, and intestinal epithelial cells on a scale from 0 to 3 (0 = 0–1 dots, 1 = rare staining but at least 2 dots, 2 = consistent, distinguishable dots throughout the section, 3 = intense staining with overlapping dots). Positive (PPIB) and negative (DapB) control probes were stained in parallel for all experiments, and six mock mice were stained.
Roth A.N., Helm E.W., Mirabelli C., Kirsche E., Smith J.C., Eurell L.B., Ghosh S., Altan-Bonnet N., Wobus C.E, & Karst S.M. (2020). Norovirus infection causes acute self-resolving diarrhea in wild-type neonatal mice. Nature Communications, 11, 2968.
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10

Localization of CXCL10-Expressing Cells in Tissues

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To localize CXCL10 positive cells, tissues were analyzed with RNAscope assay (Advanced Cell Diagnostics, Newark, CA, USA) using RNAscope 2.5 HD Assay-RED kit and Hs-CXCL10-C2 probe (Cat No. 311851-C2) recognizing the nt 2 to 1,115 of the CXCL10 reference sequence NM_001565. The sections from fixed human tissue blocks were treated following the manufacturer’s instructions. Briefly, freshly cut 3 μm sections were deparaffinized in xylene and treated with the peroxidase block solution for 10 min at room temperature followed by the retrieval solution for 15 min at 98°C and by protease plus at 40°C for 30 min. Control probes included Hs-POLR2a-C2 (Cat No. 310451) and DapB-C2 (Cat No. 310043-C2). The hybridization was performed for 2 h at 40°C. The signal was revealed using RNAscope 2.5 HD Detection Reagent and FAST RED. Combined RNAscope and immunohistochemistry (for CD163, IRF1, Phospho-STAT1, CSFR1 and TREM2) were used to identify the cellular source of CXCL10. To this end, CXCL10 detection by RNAscope was followed by immunoreaction was visualized using Novolink Polymer (Leica Microsistem) followed by DAB or using Mach 4 MR-AP (Biocare Medical) followed by Ferangi Blue (Biocare Medical).
Ardighieri L., Missale F., Bugatti M., Gatta L.B., Pezzali I., Monti M., Gottardi S., Zanotti L., Bignotti E., Ravaggi A., Tognon G., Odicino F., Calza S., Missolo-Koussou Y., Ries C.H., Helft J, & Vermi W. (2021). Infiltration by CXCL10 Secreting Macrophages Is Associated With Antitumor Immunity and Response to Therapy in Ovarian Cancer Subtypes. Frontiers in Immunology, 12, 690201.
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