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RNAscope kit

Manufactured by Advanced Cell Diagnostics
Sourced in United States

The RNAscope kit is a specialized tool designed for the detection and visualization of RNA molecules within biological samples. It utilizes an advanced in situ hybridization technology to enable the highly sensitive and specific localization of target RNA sequences in cells and tissues.

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59 protocols using RNAscope kit

1

Multiplex In Situ Hybridization and Immunohistochemistry

Tissue was embedded in Optimal Cutting Temperature medium (Sakura Tissue-Tek) and was brought to cryostat temperature (−20° C) before cutting. Chuck temperature was raised to −12°/ −10°C for optimal cutting conditions. Tissue was sectioned at 16 µm thickness onto Fisher SuperFrost slides. Direction of tissue orientation relative to the depth of the cortical sulcus was randomized across samples. Sections were fixed in cold 4°C 10% Neutral Buffered Formalin for 60 minutes and dehydrated in 50%, 70%, 100%, and 100% ethanol for 5 minutes each at room temperature. Fluorescent in situ hybridization was performed using RNAScope kits (Advanced Cell Diagnostics) optimized on the Leica BOND Rx automated slide staining system. Slides were pretreated with protease for 15 minutes. Opal TSA dyes were used for visualization at a concentration of 1:300–500. A positive and negative control probe was run for each block before staining with targeted probes. For immunohistochemical codetection of p-tau, sections were run through the RNAScope protocol as described and then manually stained with the immunohistochemical protocol described in the Histological Analysis section.
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We used RNAscope probes (Ccl2 311791; Tubb3 423391-C4) designed by Advanced Cell Diagnostics (ACD), and stained four 16-μm cryosections from the spinal cord of mice with ACD RNAscope kits according to manufacturer’s instructions.
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In situ hybridization was performed using the RNA scope kit (Advanced Cell Diagnostics) following the manufacturer’s instructions. Procr probes were ordered from Advanced Cell Diagnostics (REF#410321).
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In situ detection of GDF-11 and
GDF-15 mRNA was performed by a manual method using the
RNAscope kit (Advanced Cell Diagnostics) according to the
manufacturer’s instructions. 10 μm OCT-frozen tissue (E12,
E14, E18 and P0) sections were pretreated with hydrogen peroxide, antigen
retrieval, and protease application prior to hybridization with a target
probe to GDFs. Incubations proceeded with 30 min at
40°C; 15 min at 40°C; 30 min at 40°C; 15 min at
40°C; 30 min at room temperature; and 15 min at room temperature,
followed by washing the sections for 2 min in wash buffer between each
incubation. Colorimetric substrate was added to sections and incubated for
another 10 min at room temperature. A positive stain was indicated by red
punctate dots present in the tissue. Multiple tissues were tested and
individual representative sections shown. Three independent replications
were conducted in the study.
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Formalin-fixed mouse liver tissue from five mice (three diseased and two non-diseased) was routinely processed using haematoxylin and eosin-stained sections for examination by light microscopy. Sections from three mice (two diseased and one non-diseased liver) were also processed for fluorescent microscopy using DAPI to highlight cell nuclei and ISH to assess cell tropism for FULV and AMSV using the ViewRNA ISH Tissue 1-Plex assay (Affymetrix, Santa Clara, CA, USA). The branched DNA method employed in the ViewRNA ISH kit is similar to the RNAScope kit (Advanced Cell Diagnostics, Hayward, CA, USA) and is capable of detecting low copy number targets within formalin-fixed paraffin-embedded samples [16 (link)]. We employed a 10 min heat pretreatment and 20 min protease digestion and otherwise followed assay guidelines. Slides were mounted with VECTASHIELD HardSet Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA, USA). ISH probes were designed in the RdRp region from each viral genome. Mouse beta-actin and polyomavirus (NJPyV-2013) probes were included as controls. To estimate the percentage of infected nematode eggs, we counted the number of eggs demonstrating characteristic fluorescence using either FULV or AMSV probes. Estimates were generated using a single section of a highly infected liver.
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In situ hybridization was performed using the RNA scope kit (Advanced Cell Diagnostics) following the manufacturer’s instructions. Procr probes (REF#410321) and Ccn1 probes (REF#429001) were ordered from Advanced Cell Diagnostics. After in situ hybridization, TSA method was used for Krt8 staining following the manufacturer’s instructions using the Opal 4-Color Automation IHC Kit (PerkinElmer). The images were captured using Leica DM6000 TCS/SP8 laser confocal scanning microscope with a ×63/0.75 IMM objective.
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Detection of LGR5 mRNA was performed with an RNAscope® kit (Advanced Cell Diagnostics, Hayward, CA, USA) according to the manufacturer’s instructions using unstained sample tissue slides. Briefly, tissue sections were pretreated by heating and protease was applied prior to hybridization with an LGR5-specific probe. The detailed procedure was described in a previous publication [16 (link)]. Brown dots present in the nucleus and/or cytoplasm were recognized as positive staining. LGR5 expression was quantified using a five-level scoring system recommended by the manufacturer (0, no staining; 1, 1–3 dots/cell; 2, 4–10 dots/cell; 3, > 10 dots/cell; 4, > 15 dots/cell with > 10% of dots in clusters). The H-score was calculated as: (% of grade 1 cells × 1) + (% of grade 2 cells × 2) + (% of grade 3 cells × 3) + (% of grade 4 cells × 4). The overall H-score for each patient was calculated based on the H-score per high-power field (400× magnification). Furthermore, any cell with one or more dots was regarded as LGR5-positive.
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8

Immunohistochemistry and RNA in situ Hybridization of Inflammatory Markers

For immunohistochemistry (IHC), sectioned formalin-fixed paraffin-embedded biopsy samples were deparaffinized and rehydrated, which was followed by heat-induced and enzymatic antigen retrieval. Subsequently, sections were blocked and incubated at 4 °C overnight with the following primary antibodies: anti-IL-1β antibody (1:100, 12242; Cell Signaling Technology, Inc., Danvers, MA, USA) or anti-IL-18 antibody (1:1000, ab243091; Abcam, Cambridge, UK). Thereafter, biotinated or horseradish peroxidase-conjugated secondary antibodies (HAF007; R&D Systems, Inc., Minneapolis, MN, USA) were used for detection. Anti-CD66b antibodies (1:100, 3929023, Biolegend, San Diego, CA, USA) were used for the detection of neutrophils. Images were captured using Axio Scope AI (ZEISS, Oberkochen, Germany).
For RNA in situ hybridization, the RNAScope Kit (Advanced Cell Diagnostics, Newark, CA, USA) was used to measure the mRNA expression in paraffin-embedded sections.
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In situ hybridization for Olfm4 mRNA was performed using the RNAscope kit (Advanced Cell Diagnostics) according to the manufacturer’s instructions. Briefly, 5 μm formalin-fixed, paraffin embedded tissue sections or 8 μm OCT frozen sections were pretreated with heat and protease prior to hybridization with a target probe to Olfm4 mRNA. An HRP-based signal amplification system was then hybridized to the target probes followed by colorimetric development with DAB. Negative control probes for the bacterial gene DapB were also included for each slide.
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10

In Situ Hybridization for Neuropeptide and Neurotransmitter Markers

Tissue was processed using in situ hybridization to detect mRNA for Nts, Vgat (Slc32a1), and Vglut2 (Slc17a6). Mice were briefly anesthetized with 3.5 – 4.0% isoflurane mixed with pure oxygen (1L/min), rapidly decapitated and brains rapidly extracted and flash frozen on dry ice. 18-μm thick cryostat coronal sections were collected under RNAse-free conditions, fixed in 4% PFA for 15 min at 4°C, dehydrated in serial concentrations of ethanol (50%–100%), and processed according to the protocol provided in the RNAscope kit (Advanced Cell Diagnostics. INC, Cat. 320 293). Sections were hybridized with the following mixed probes; Nts (Mm-Nts, Cat. 420441), Vgat (Mm-Slc32a1, Cat. 319191), and Vglut2 (Mm-Slc17a6-C2, 319171), for 2 h at 40°C and following amplification, sections were counterstained with DAPI. For Nts and Cre, were performed in situ using the Affymetrix View RNA 2-Plex Tissue Assay Kit with custom probes for Nts (Mouse NM024435, Cat. VB1-16908) and Cre (Vector, HQ335171 Cat.designed by Affymetrix (Santa Clara, CA).
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