Log In Sign up
The largest database of trusted experimental protocols

Rnascope 2.5 hd assay

Manufactured by Advanced Cell Diagnostics
Visit Supplier
Sourced in United States

The RNAScope 2.5 HD Assay is a multiplex fluorescent in situ hybridization (FISH) technology developed by Advanced Cell Diagnostics. The assay enables the detection and quantification of target RNA molecules within intact cells and tissues, allowing for the visualization and analysis of gene expression patterns at a single-cell level.

Automatically generated - may contain errors

Lab products found in correlation

Imaris v9, by Oxford Instruments (2 mentions) Eclipse ni microscope, by Nikon (2 mentions) Ecomount, by Biocare Medical (1 mentions) Ndp view2, by Hamamatsu Photonics (1 mentions) Hematoxylin, by Merck Group (1 mentions) Horseradish peroxidase conjugated streptavidin, by Thermo Fisher Scientific (1 mentions) Nanozoomer 2.0 ht, by Hamamatsu Photonics (1 mentions) Vectastain abc kit, by Vector Laboratories (1 mentions) Dab peroxidase substrate kit, by Vector Laboratories (1 mentions) Anti ifn α, by PBL Assay Science (1 mentions) Anti cd31 jc70a, by Agilent Technologies (1 mentions)

12 protocols using rnascope 2.5 hd assay

1

In Situ Hybridization of Mouse Eye Tissue

Check if the same lab product or an alternative is used in the 5 most similar protocols
One-month-old mice (C57/BL6J or BALB/cJ) were euthanized by CO2 inhalation. Eye tissue was fixed overnight in 4% paraformaldehyde/PBS and dehydrated through a graded series of ethanols and xylene. Tissue was embedded in paraffin and sectioned in the midsagittal plane at a thickness of 4 μm. Expression of selected genes was visualized by in situ hybridization using the RNAscope technique (RNAscope 2.5 HD assay; Advanced Cell Diagnostics, Hayward, CA), as described (Shi et al., 2013 (link); Wang et al., 2012 (link)). Target probe sets are listed in Table 1. Probes consisted of 20 pairs of oligonucleotides spanning a ≈1 kb region of the target mRNA transcript. Following proprietary preamplification and amplification steps, binding was visualized using an alkaline-phosphatase-conjugated probe with Fast Red as a substrate. As a negative control, adjacent tissue sections were incubated with a probe against DapB, a bacterial gene. The ubiquitously expressed PolR2A (DNA-directed RNA polymerase II polypeptide A) was used as a positive control. Results are representative of at least three independent experiments in each case. All in situ hybridization experiments were performed on both BALB/cJ and C57/BL6J mice with comparable results.
Youkilis J.C, & Bassnett S. (2021). Single-cell RNA-sequencing analysis of the ciliary epithelium and contiguous tissues in the mouse eye. Experimental eye research, 213, 108811.
+ Open protocol
+ Expand
2

In-Situ Hybridization of Varicella-Zoster Virus

Cited 2 times
Check if the same lab product or an alternative is used in the 5 most similar protocols
ISH was performed using the RNAScope 2.5 HD Assay and probes directed to SVV VLT (core VLT exons 1–3; cat# 549461), SVV ORF63 (cat# 438091), universally expressed positive control gene ubiquitin C (UBC) and negative control bacterial transcript DapB (all from Advanced Cell Diagnostics). Staining was visualized using FastRed as a substrate, nuclei were stained with hematoxylin and slides were mounted with Ecomount (Biocare Medical). ISH was performed on normal skin and varicella skin rash of n = 2 animals (AGM 269 and 279 in [23 (link)]), with n = 2 independent experiments and 3–4 skin biopsies per tissue section. Additionally, we performed ISH on 21 DRG from n = 2 animals (RM 2207 and RM 9021, [40 (link)]), encompassing 3 or 4 sacral ganglia and 7 lumbar ganglia per animal.
Braspenning S.E., Verjans G.M., Mehraban T., Messaoudi I., Depledge D.P, & Ouwendijk W.J. (2021). The architecture of the simian varicella virus transcriptome. PLoS Pathogens, 17(11), e1010084.
+ Open protocol
+ Expand
3

Detecting Latent VZV Infection via IHC and ISH

Cited 2 times
Check if the same lab product or an alternative is used in the 5 most similar protocols
Consecutive 5 µm-thick FFPE TG sections from five latent VZV-infected subjects (Additional file 4: Table S2) were analyzed for CD3 protein expression and VZV VLT RNA expression by immunohistochemistry (IHC) and in situ hybridization (ISH), respectively, as described previously [3 (link), 24 (link)]. In brief, IHC was performed using mouse anti-CD3 antibody (clone F7.2.38), biotin-conjugated goat anti-mouse IgG antibody, and horseradish peroxidase-conjugated streptavidin (all from ThermoFisher). Staining was visualized using 3-amino-9-ethylcarbazole (AEC) and sections were counterstained with hematoxylin (Sigma-Aldrich). ISH was performed using probes specific to VZV VLT [3 (link)], mRNA of the human gene POLR2A (encoding DNA-directed RNA polymerase II subunit RPB1; positive control), and the bacterial gene dapB (encoding 4-hydroxy-tetrahydrodipicolinate reductase, Genbank Accession number EF191515; negative control) using the RNAScope 2.5 HD Assay (Advanced Cell Diagnostics). RNA integrity in tissue sections and specificity of the RNAScope Assay was demonstrated by robust POLR2A staining and absence of dapB ISH signal in all sections analyzed (Additional file 1: Figure S1). Complete tissue sections were scanned using the Nanozoomer 2.0 HT (Hamamatsu) and analyzed using NDP.view2 (Hamamatsu).
van Gent M., Ouwendijk W.J., Campbell V.L., Laing K.J., Verjans G.M, & Koelle D.M. (2023). Varicella-zoster virus proteome-wide T-cell screening demonstrates low prevalence of virus-specific CD8 T-cells in latently infected human trigeminal ganglia. Journal of Neuroinflammation, 20, 141.
+ Open protocol
+ Expand
4

Multiplexed ISH of Placental and Liver Genes

Check if the same lab product or an alternative is used in the 5 most similar protocols
ISH was performed using the RNAscope 2.5 HD Assay (322310 and 322360; Advanced Cell Diagnostics, Newark, CA) and appropriate probes and as per the manufacturer’s directions. The following probes were used: Ascl1 (476321; Advanced Cell Diagnostics), Igf2 (437671; Advanced Cell Diagnostics), PL-I (405521; Advanced Cell Diagnostics), and PL-II (423681; Advanced Cell Diagnostics). A positive control probe Ppib (310043; Advanced Cell Diagnostics) and a negative control probe DapB (313911; Advanced Cell Diagnostics) were used to determine the efficacy of the protocol. These probes were applied to formalin-fixed, paraffin-embedded sections of mouse livers and placentas. The slide images were acquired by the Leica DM2000 microscope using the Leica Application Suite program. After approval from the institutional review boards, the laboratory information systems of our institute were searched. A set of paraffin blocks archived in the Department of Pathology of Indiana University School of Medicine were selected. They represented liver tissues from pregnant patients and patients with hepatocellular carcinoma or hepatocellular adenoma. These paraffin blocks were sectioned for ISH with a human Ascl1 probe (459721; Advanced Cell Diagnostics).
Lee J., Garcia V., Nambiar S.M., Jiang H, & Dai G. (2021). Activation of Proneuronal Transcription Factor Ascl1 in Maternal Liver Ensures a Healthy Pregnancy. Cellular and Molecular Gastroenterology and Hepatology, 13(1), 35-55.
+ Open protocol
+ Expand
5

HIV-1 RNA Expression in Prefrontal Cortex

Cited 1 time
Check if the same lab product or an alternative is used in the 5 most similar protocols
Paraffin-embedded sections of prefrontal cortex (from the same subjects) were provided by the NNTC upon request. RNAScope 2.5 HD assay (Advanced Cell Diagnostics, ACD) was performed. Briefly, pre-treatment was performed with RNAscope 2.5 HD Detection Kit (RED) (Cat# 322360), RNAscope 2.5 Pretreat Reagents-H202 and Protease Plus (Cat#322330), RNAscope Target Retrieval (Cat#322000) and RNAscope Wash Buffer (Cat#310091), following manual instructions. Probe set V-HIV1-clade B-C3 (Cat#425531-C3) targeting different segments within the gag-pol region, as described [31 (link),32 (link),33 (link)]. Images were captured using the light feature of a Zeiss AXIO Observer.Z1 (Carl Zeiss AG, Oberkochen, Germany).
Basova L., Lindsey A., McGovern A.M., Ellis R.J, & Marcondes M.C. (2021). Detection of H3K4me3 Identifies NeuroHIV Signatures, Genomic Effects of Methamphetamine and Addiction Pathways in Postmortem HIV+ Brain Specimens that Are Not Amenable to Transcriptome Analysis. Viruses, 13(4), 544.
+ Open protocol
+ Expand
6

Immunohistochemistry and RNA in situ Hybridization

Check if the same lab product or an alternative is used in the 5 most similar protocols
Formalin-fixed paraffin-embedded tissue sections were stained by standard immunohistochemistry using a VECTASTAIN ABC kit (Vector Laboratories) and DAB Peroxidase Substrate kit (Vector Laboratories) according to the manufacturer’s instructions. For RNA in situ hybridization, 5-µm formalin-fixed, paraffin-embedded tissue sections were processed using the RNAscope 2.5 HD Assay and Mm-Ulbp1 probe (target region 2–1,472; Advanced Cell Diagnostics) according to the manufacturer’s instructions. The Ulbp1+ signals by in situ hybridization were counted as pixels per high-power field using ImageJ software (National Institutes of Health).
Hosomi S., Grootjans J., Tschurtschenthaler M., Krupka N., Matute J.D., Flak M.B., Martinez-Naves E., Gomez del Moral M., Glickman J.N., Ohira M., Lanier L.L., Kaser A, & Blumberg R. (2017). Intestinal epithelial cell endoplasmic reticulum stress promotes MULT1 up-regulation and NKG2D-mediated inflammation. The Journal of Experimental Medicine, 214(10), 2985-2997.
+ Open protocol
+ Expand
7

Comprehensive FFPE Tissue Analysis

Cited 1 time
Check if the same lab product or an alternative is used in the 5 most similar protocols
FFPE tissue sections were analyzed by H&E staining, IHC, immunofluorescence (IF), and ISH. IHC and IF was performed using the following primary antibodies: mouse anti-CD3 (clone F7.2.38), anti-CD20 (L26), anti-CD68 (KP1), anti-granzyme B (GrB-7), anti–neutrophil elastase (NP57), anti–CD45 (2B11 + PD7/26), and anti-CD31 (JC70A) (all from Dako); anti-cytokeratin (AE1/AE3) and anti-pSTAT1 (Tyr701; clone ST1P-11A5) (Thermo Fisher Scientific); goat anti-MPO (AF3667; R&D systems); rabbit anti-citH3 (ab5103), anti–claudin-18 (34H14L15), and anti–VE cadherin (ab33168) (Abcam); and anti–IFN-α (PBL Assay Science) as described in Supplemental Methods. ISH was performed using probes directed to SVV ORF63 (71 (link)), CCL2, CXCL8, CXCL10, ubiquitin C (positive control), and the bacterial gene dapB (negative control) using the RNAScope 2.5 HD Assay (Advanced Cell Diagnostics) (Supplemental Methods).
Ouwendijk W.J., van den Ham H.J., Delany M.W., van Kampen J.J., van Nierop G.P., Mehraban T., Zaaraoui-Boutahar F., van IJcken W.F., van den Brand J.M., de Vries R.D., Andeweg A.C, & Verjans G.M. (2020). Alveolar barrier disruption in varicella pneumonia is associated with neutrophil extracellular trap formation. JCI Insight, 5(21), e138900.
+ Open protocol
+ Expand
8

Quantitative smFISH of Cochlear Genes

Check if the same lab product or an alternative is used in the 5 most similar protocols
Single molecule fluorescence in situ hybridization (smFISH) was used to examine the expression of 10 genes in 10 mm-thin cochlear sections. The cochlea was prepared as described above for immunocytochemistry. Samples were prepared in PFA-fixed paraffin embedded tissue. Methods for the RNAscope™ 2.5 HD Assay from Advanced Cell Diagnostics were followed. Probes for 12 genes were purchased from ACD (Tmc1 (Cat No: 520911-C2), Chrna9 (818511), Kcnq4 (707481), Chrna10 (818521), Slc17a8 (549991), Slc7a14 (544781), Otof (485678), Clu (427891), Jund (1031951-C1), Foxo3 (485321), C1ql1 (465081-C2), and Sod1 (428581)). To compare the expression of gene of interest between 9 and 26 m, intensity (gray value) and area of the fluorescent signals were measured to obtain integrated density for individual IHCs and OHCs in each cochlear section. Fluorescent signals from three sections were averaged for each cell type at each age.
Liu H., Giffen K.P., Chen L., Henderson H.J., Cao T.A., Kozeny G.A., Beisel K.W., Li Y, & He D.Z. (2022). Molecular and cytological profiling of biological aging of mouse cochlear inner and outer hair cells. Cell reports, 39(2), 110665.
+ Open protocol
+ Expand
9

In Situ Hybridization of Mouse Eye Tissue

Check if the same lab product or an alternative is used in the 5 most similar protocols
One-month-old mice (C57/BL6J or BALB/cJ) were euthanized by CO2 inhalation. Eye tissue was fixed overnight in 4% paraformaldehyde/PBS and dehydrated through a graded series of ethanols and xylene. Tissue was embedded in paraffin and sectioned in the midsagittal plane at a thickness of 4 μm. Expression of selected genes was visualized by in situ hybridization using the RNAscope technique (RNAscope 2.5 HD assay; Advanced Cell Diagnostics, Hayward, CA), as described (Shi et al., 2013 (link); Wang et al., 2012 (link)). Target probe sets are listed in Table 1. Probes consisted of 20 pairs of oligonucleotides spanning a ≈1 kb region of the target mRNA transcript. Following proprietary preamplification and amplification steps, binding was visualized using an alkaline-phosphatase-conjugated probe with Fast Red as a substrate. As a negative control, adjacent tissue sections were incubated with a probe against DapB, a bacterial gene. The ubiquitously expressed PolR2A (DNA-directed RNA polymerase II polypeptide A) was used as a positive control. Results are representative of at least three independent experiments in each case. All in situ hybridization experiments were performed on both BALB/cJ and C57/BL6J mice with comparable results.
Youkilis J.C, & Bassnett S. (2021). Single-cell RNA-sequencing analysis of the ciliary epithelium and contiguous tissues in the mouse eye. Experimental eye research, 213, 108811.
+ Open protocol
+ Expand
10

Quantifying TrkB mRNA Expression in Striatal Tissue

Check if the same lab product or an alternative is used in the 5 most similar protocols
To examine the impact of rs6265 on host striatal mRNA for the BDNF
receptor, tyrosine receptor kinase B (TrkB), we performed in
situ hybridization (ISH) for Trkb mRNA on
1–2 tissue sections per animal at 40-μm thickness using the
manual RNAscope® 2.5 HD assay (Advanced Cell Diagnostics Inc.,
Hayward, CA, USA) according to manufacturer instructions, then
counterstained with cresyl violet. Images (2880 × 2048) were acquired
in the dorsolateral striatum adjacent to the graft using the 20x objective
on a Nikon Eclipse Ni microscope, maintaining identical light settings
across all images. The images were imported into the image visualization and
analysis software, Imaris® (v. 9.3.1, Oxford Instruments) using the
ImageJ (FIJI) extension. The Imaris® spots function was used to
reconstruct Trkb mRNA puncta in a two-dimensional field of
view, using the same parameters for all images, and the resulting data were
exported. Data are represented as the average of values collected from two
to four images per striatal hemisphere.
Mercado N.M., Stancati J.A., Sortwell C.E., Mueller R.L., Boezwinkle S.A., Duffy M.F., Fischer D.L., Sandoval I.M., Manfredsson F.P., Collier T.J, & Steece-Collier K. (2020). The BDNF Val66Met polymorphism (rs6265) enhances dopamine neuron graft efficacy and side-effect liability in rs6265 knock-in rats. Neurobiology of disease, 148, 105175.
+ Open protocol
+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required

Sign up now

Revolutionizing how scientists
search and build protocols!