RNA in-situ hybridization was performed using BaseScope Detection Reagent v2-RED Assay Kit (Advanced Cell Diagnostics, Inc. #323900), following manufacturer’s instructions. Expression of transcripts containing cryptic exon splice sites in Unc13a and Ift81 transcripts was detected using 3zz custom BaseScope probes (BA-Mm-Unc13a-O1-2EJ-C and BA-Mm-Ift81-E17-intron17-NJ, respectively). Both positive and negative control probes were employed to assess the RNA quality (BA-Mm-Ppib and BA-DapB). Briefly, consecutive tissue sections of 10 μM thickness underwent de-paraffinization followed by pre-treatment with hydrogen peroxide, target retrieval buffer, and protease IV. Subsequently, these sections were subject to hybridization with target probes in a HybEZII oven (Advanced Cell Diagnostics, Inc.) for 2 hours at 40°C. The signals were amplified, and the slides were counterstained with hematoxylin. Images were acquired using a Zeiss Apotome Inverted Brightfield Microscope (Zeiss, Germany), and RNA puncta for each cryptic exon were manually quantified using ImageJ software.
Hybez 2 oven
Manufactured by Advanced Cell Diagnostics
The HybEZ™ II oven is a specialized laboratory equipment designed for precise temperature control during in situ hybridization and immunohistochemistry procedures. It provides a consistent and regulated environment to facilitate optimal reaction conditions for these critical analytical techniques.
Automatically generated - may contain errors
Lab products found in correlation
Immedge hydrophobic barrier pen, by Vector Laboratories (2 mentions)
Rnascope antigen retrieval reagent, by Advanced Cell Diagnostics (2 mentions)
Rnascope protease plus reagent, by Advanced Cell Diagnostics (2 mentions)
Superfrost plus, by Thermo Fisher Scientific (2 mentions)
Hydrogen peroxide, by Advanced Cell Diagnostics (1 mentions)
Hybez 2, by Advanced Cell Diagnostics (1 mentions)
V ncov2019 s probe, by Advanced Cell Diagnostics (1 mentions)
Gibco dpbs, by Thermo Fisher Scientific (1 mentions)
Rnascope multiplex fluorescent reagent kit v2 assay, by Advanced Cell Diagnostics (1 mentions)
4 protocols using hybez 2 oven
1
Detecting Cryptic Exons in Unc13a and Ift81
2
In Situ mRNA Detection in FFPE Fish Tissues
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Formalin fixed, paraffin embedded (FFPE) tissue sections (5 µm thickness) from heart and skeletal muscle tissues selected from fish with HSMI, were mounted using Superfrost plus (Thermo Fisher Scientific) slides. Sections were baked at 60°C for 2 hrs in HybEZ™ II oven (Advanced Cell Diagnostics, catalog #321720) prior to deparaffinization with absolute ethanol (100%) and fresh xylene. Initial blocking was done with hydrogen peroxide (Advanced Cell Diagnostics) for 10 min at room temperature (RT). RNAscope antigen retrieval reagent (Advanced Cell Diagnostics, catalog #322000) was used for 15 min at 99°C and slides were further incubated with RNAscope protease plus reagent for 15 min at 40°C in the HybEZ™ II oven following manufacturer guidelines. Immedge hydrophobic barrier pen (Vector Laboratories, Burlingame, CA) was used to make hydrophobic barrier around tissue areas over slides for further probe hybridization procedures.
3
RNAscope Sample Preparation
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FFPE sections were sliced with 5 µm thickness from tissue samples and mounted on Superfrost plus (Thermo Fisher Scientific) slides. Slides were baked at 60°C for 2 h in a HybEZ™ II oven (Advanced Cell Diagnostics, catalog #321720) followed by deparaffinization with 100% ethanol and fresh xylene baths. Samples were pretreated with hydrogen peroxide for 10 min at RT, boiled with RNAscope antigen retrieval reagent (Advanced Cell Diagnostics, catalog #322000) for 15 min at 99°C, and then incubated with RNAscope protease plus reagent for 15 min at 40°C in the HybEZ™ II oven. Hydrophobic barrier was made around the tissue section using Immedge hydrophobic barrier pen (Vector Laboratories, Burlingame, CA).
4
SARS-CoV-2 S-Protein Expression in Post-Mortem Brains
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Expression of the SARS-Cov2 S-protein in post mortem brains was assessed using an RNAscope® Multiplex Fluorescent Reagent kit v2 Assay and the V-nCov2019-S probe, reference: 848,561 (both from Advanced Cell Diagnostics Inc.). Briefly, 20 μm-thick hypothalamic sections were cut on a cryostat, slides washed twice for 10 min in Gibco® DPBS (ThermoFisher) and dry baked in a HybEZ™ II oven (Advanced Cell Diagnostics Inc.) at 60 °C for 30 min. They were then immersion-fixed in 4% paraformaldehyde PBS 0.1 M, pH 7.4, prepared in DEPC-treated water, for 1 h at 4 °C and washed again twice for 10 min in Gibco® DPBS. Next, the sections were processed according to manufacturer's instructions (ethanol dehydration, RNAscope hydrogen peroxide treatment and target retrieval), incubated with RNAscope protease IV for 10 min at room temperature, and the signal revealed using the RNAscope multiplex fluorescent assay.
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