mRNA was detected in sections using the manual RNAScope 2.5 HD BROWN assay or, for dual in situ hybridisation, the RNAScope 2.5 HD Duplex assay (Advanced Cell Diagnostics, Newark, US). RNAScope probes: Mm-Pnmt (426421 or 426421-C2 for dual RNAScope), Mm-Epas1 (314371), Mm-Rgs5 (430181), Mm-Vegfa-OI (43961). Imaging was performed with a Leica DM 1000 LED microscope (Leica Biosystems).
Rnascope 2.5 hd assay brown
Manufactured by Advanced Cell Diagnostics
Sourced in United States
RNAscope 2.5 HD Assay-Brown is an in-situ hybridization technology developed by Advanced Cell Diagnostics. The assay enables the detection and quantification of target RNA molecules within intact cells and tissues.
Automatically generated - may contain errors
Lab products found in correlation
Dnase 1, by Thermo Fisher Scientific (6 mentions)
Rnascope ez batch slide processing system, by Advanced Cell Diagnostics (2 mentions)
Rnascope probe mm lta, by Advanced Cell Diagnostics (2 mentions)
Dm 1000 led microscope, by Leica (1 mentions)
Rnascope 2.5 hd duplex assay, by Advanced Cell Diagnostics (1 mentions)
Aperio at2 digital slide scanner, by Leica (1 mentions)
Nb300 109, by Novus Biologicals (1 mentions)
Imagescope software, by Leica (1 mentions)
At2 slide scanner, by Leica (1 mentions)
Rnase t1, by Thermo Fisher Scientific (1 mentions)
Dnase 1, by Qiagen (1 mentions)
Rnase a, by Qiagen (1 mentions)
Bx53m microscope, by Olympus (1 mentions)
Rnascope spotstudio v1, by Advanced Cell Diagnostics (1 mentions)
Superfrost plus glass slide, by Thermo Fisher Scientific (1 mentions)
Axioimager d1 fluorescent microscope, by Zeiss (1 mentions)
Zinc formalin fixative, by Polysciences (1 mentions)
Uplansapo 20x 0, by Olympus (1 mentions)
Aperio cs2, by Leica (1 mentions)
Axio imager m2 imaging system, by Zeiss (1 mentions)
32 protocols using rnascope 2.5 hd assay brown
1
mRNA Detection in Tissue Sections
2
In Situ Hybridization of HCV Genotypes
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In situ hybridization assays on liver biopsies were performed using the RNAscope® 2·5 HD-Brown assay (Advanced Cell Diagnostics, Inc., Newark, CA, USA). The detailed information of each probe was: RNAscope® Probe- V-HCV-GT1 (Cat#423221, target region: 36–2399), RNAscope® Probe- V-HCV-GT2 (Cat#423231, target region: 19–2439), RNAscope® Probe- V-HCV-GT3 (Cat#423241, target region: 178–2454), RNAscope® Probe- V-HCV-GT4 (Cat#423251, target region: 103–2529), RNAscope® Probe- V-HCV-GT5 (Cat#423251, target region: 2–2896), RNAscope® Probe- V-HCV-GT6 (Cat#423271, target region: 2–2623), RNAscope® Positive Control Probe-Hs-PPIB (Cat#313901, target region: 139–989 of NM_000942·4), and RNAscope® Negative Control Probe-DapB (Cat#310043, target region: 414–862 of EF191515). The images were acquired using an Aperio AT2 digital slide scanner equipped with a 40× objective (Leica Biosystems Inc., Buffalo Grove, IL, USA). The performance procedure was provided in supplementary information.
3
In situ SHIV RNA and DNA Localization
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In situ hybridization of SHIV viral RNA and integrated proviral DNA was performed using the RNAscope 2.5 HD Brown Assay (Advanced Cell Diagnostics) with the SIVmac239 probe (catalog 312811) and the SIVmac239-sense probe (catalog 314071), respectively. Photomicrograph images were taken with a Nikon E800 at 20× and 40× objectives.
4
RNA in situ Hybridization of LINC01187
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RNA in situ hybridization (RNA-ISH) was performed using the RNAscope 2.5 HD Brown assay (Advanced Cell Diagnostics, Newark, CA) and target probes against LINC01187 (catalog no. 532311) as previously described (10 (link)). Dual RNA-ISH was performed using the RNAscope 2.5 HD duplex assay and target probes against L1CAM, LINC01187, and FOXI1 (catalog no. 567451,532311-C2, and 476359-C2). RNA quality was evaluated using positive control probe targeting human housekeeping gene PPIB. All evaluated cases passed RNA-ISH quality control (QC) except one ESC-RCC case. Assay background was monitored using a negative control probe targeting bacterial DapB gene. Stained slides were examined under 100x and 200x magnification for RNA signals in tumor cells and adjacent benign kidney tissues. The RNA-ISH assay stained each RNA molecule as an individual brown, punctate dot. The staining was independently assessed by three study participants including two pathologists (XWang, RMannan, and RMehra) at 100x, 200x and 400x magnification to assess for presence and patterns of expression.
5
Immunohistochemical and In Situ Analysis of Carotid Body
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Paraffin-embedded sections were immunostained with anti-TH (dilution 1:5000; NB300-109; Novus Biologicals) or with an anti-BrdU antibody (dilution 1:10; catalog 551321; BD Biosciences), as described previously (25 (link)). Vegfa and Hif-2α mRNA transcripts were detected via in situ hybridization on sections from paraffin-embedded tissues using the manual RNAscope 2.5 HD BROWN assay (Advanced Cell Diagnostics). Samples were imaged using a DM 1000 LED microscope (Leica Biosystems). Stereological estimation of cell number, cell density, and carotid body volume was performed using ImageJ (NIH) on every fourth paraffin section (25 (link)). In situ hybridization probe signal was quantified using trainable Weka segmentation in FIJI ImageJ software (NIH), according to the manufacturer’s instructions.
6
Quantifying Gene Expression with RNAScope
7
In Situ RNA Hybridization of Kidney Markers
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In situ RNA hybridization was performed using RNAscope 2.5 HD–BROWN assay (Advanced Cell Diagnostics [ACD], 322300) according to the manufacturer’s protocol. Briefly, kidneys harvested from mice of ages ranging from E13 to 8 weeks were fixed in formalin, embedded in paraffin, and cut into sections of 5–20 μm. The kidney sections were deparaffinized in xylene and 100% ethanol, air dried for 5 minutes, and covered by 3% hydrogen peroxide for 10 minutes. After target retrieval for 15 minutes at 95°C, the kidney slides were digested by Protease Plus for 30 minutes at 40°C and hybridized with Clc-k1 or Clc-k2 probes for 2 hours at 40°C. The slides were then washed by a 1× wash buffer following each amplification step. The RNA signals were revealed by incubating slides with BROWN-A and -B solution for 10–60 minutes, counterstaining with 12.5% hematoxylin, and mounting with Sub-X medium (Leica Biosystems, 3801740). The following RNAscope probes were custom designed by ACD: Clcnka (NM_001146307.1, region 2-2389; 536031), Clcnkb (NM_019701.2, region 591-2341; 528541).
8
Detecting Plekhg5 mRNA in Mouse Tissue
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The RNAscope 2.5 HD Brown assay (Advanced Cell Diagnostics) was used to detect mRNAs in 5 μm mouse tissue sections. In situ hybridization was performed according to the manufacturer’s protocol with the Hs-Plekhg5 probe (NM_001042663.1, target region 689–1,750, catalog 415321). Images were captured using an AT2 slide scanner and ImageScope software (Leica Biosystems).
9
Quantifying NNT-AS1 Expression in HCC TMA
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The expression of NNT-AS1 was detected in HCC TMA slides by an RNAscope assay kit (RNAscope® 2.5 HD Assay-Brown, Advanced Cell Diagnostics, Hayward, CA, USA, Cat No. 322310). A RNAscope probe targeting NNT-AS1 was obtained from ACD (Advanced Cell Diagnostics, Cat No. 17268B). NNT-AS1 RNA molecules were detected with single-copy detection sensitivity in TMA tissues. To calculate the number of NNT-AS1 RNA molecules, the single-molecule signals were quantified on a cell-by-cell basis by manual counting. The signals per cell were divided into the following 5 levels: 0-1 dots/10 cells, 1-3 dots/cell, 4-10 dots/cell, > 10 dots/cell with dots in clusters and positive cells in clusters < 10%, and > 10 dots/cell with dots in clusters and positive cells in clusters > 10%. To explain more conveniently,these values were marked as -, +, ++, +++, and ++++, respectively [28] . We used (Advanced Cell Diagnostics; catalog number 476701), a probe targeting human PPIB mRNA, as a positive control. A probe targeting Bacillus subtilis DapB mRNA (Advanced Cell Diagnostics; Cat No. 310043) was used as the negative control.
10
Detecting MmuPV1 Viral Transcripts using RNAscope
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MmuPV1 viral transcripts were detected using RNAscope 2.5 HD Assay-Brown (Advanced Cell Diagnostics, Newark, CA) according to the manufacturer’s instructions (61 (link)) with probes specific for MmuPV1 E1^E4 (catalog no. 473281). Tissue sections were treated following protease treatment and prior to probe hybridization with 20 units of DNase I (Thermo Fisher Scientific; catalog no. EN0521) for 30 min at 40°C. Slides were counterstained with hematoxylin before mounting and coverslipping.
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