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Rnascope protease 4

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RNAscope protease IV is a laboratory reagent used in the RNAscope assay, a technique for the detection and visualization of RNA molecules in fixed tissue samples. The core function of RNAscope protease IV is to pretreat the samples, helping to expose the RNA targets and facilitate the subsequent hybridization and detection steps of the RNAscope assay.

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Lab products found in correlation

Hybez oven, by Advanced Cell Diagnostics (3 mentions) Eclipse ti e microscope, by Nikon (2 mentions) Rnascope fluorescent multiplex reagent kit, by Advanced Cell Diagnostics (2 mentions) Hs sox2, by Advanced Cell Diagnostics (2 mentions) Hs dlx2 c2, by Advanced Cell Diagnostics (2 mentions) Hs pax6 c3, by Advanced Cell Diagnostics (2 mentions) Basescope detection reagent kit v2 red, by Advanced Cell Diagnostics (1 mentions)

4 protocols using rnascope protease 4

1

Multiprobe RNA in situ Hybridization in Forebrain Organoids

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RNA in situ hybridization was performed using the RNAscope Fluorescent Multiplex Reagent Kit (Advanced Cell Diagnostics). The samples were prepared according to the manufacturer’s instructions. Briefly, forebrain organoids were collected and embedded in OCT. The fresh frozen sample were equilibrated to −20°C in a cryostat for 1 h. 15um FFPE tissue sections were mounted on SuperFrost Plus slides, fixed in 4% PFA for 15 min at 4°C, dehydrated using 50%, 70% and 100% ethanol, and treated with RNAscope protease IV (Advanced Cell Diagnostics) for 15 min at room temperature. Probes were mixed at a 50:1:1 ratio (C1: C2:C3) and hybridized for 2 h at 40°C in the HybEZ Oven (Advanced Cell Diagnostics). The following probes from Advanced Cell Diagnostics were used: Hs-SOX2 (catalog #400871-C1), Hs-DLX2-C2 (catalog #483311-C2), and Hs-PAX6-C3 (catalog #588881-C3). Amplification and detection were performed according to the manufacturer’s instructions. Slides were applied to DAPI for 30s at room temperature, added fluorescent mounting medium and covered with coverslips. All images were captured by Nikon Eclipse Ti-E microscope. Quantitative analyses were conducted randomly on 5 regions per section in a blind fashion. The area positive for each probe was measured using ImageJ software (NIH).
Kang Y., Zhou Y., Li Y., Han Y., Xu J., Niu W., Li Z., Liu S., Feng H., Huang W., Duan R., Xu T., Raj N., Zhang F., Dou J., Xu C., Wu H., Bassell G.J., Warren S.T., Allen E.G., Jin P, & Wen Z. (2021). A human forebrain organoid model of fragile X syndrome exhibits altered neurogenesis and highlights new treatment strategies. Nature neuroscience, 24(10), 1377-1391.
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2

Gsdmc2-3 and Gsdmc4 Expression Analysis by BaseScope

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Intestinal tissue was processed for in situ hybridization using BaseScope™ Detection Reagent Kit v2-RED (323900, Advanced Cell Diagnostics) according to the manufacturer’s protocol. The test BaseScope probes 1) BA-Mm-Gsdmc2–3-zz and 2) BA-Mm-Gsdmc4-zz were ‘ZZ’ antisense probes designed to targeting mouse Gsdmc2–3 and Gsdmc4, respectively. The positive control probe is BA-Mm-Ppib-3zz and negative control probe: BA-Dapb-3zz.
Briefly, deparaffinized and dried sections were incubated with RNAscope® Hydrogen Peroxide for 10 min at room temperature and then 1xRNAscope® Target Retrieval Reagents for 15 min at 98~102°C. The samples were then incubated with RNAscope® Protease IV in HybEZ™ Oven (Advanced Cell Diagnostics, Hayward, CA) at 40°C for 30 min. Then each sample was hybridized with one BaseScope probe according to the requirement in HybEZ™ Oven at 40°C for 2 hours. After buffer washing steps, the samples were incubated in HybEZ™ Oven at 40°C with the serial application of BaseScope™ v2 Amp 1~8 for signal amplification. At last, Fast Red substrate was added to samples for 10 min at RT to detect target RNA and the slides were counterstained with 50% hematoxylin staining solution for 2 min at RT. Target RNA was visualized using a standard bright field microscope.
Zhao M., Ren K., Xiong X., Xin Y., Zou Y., Maynard J.C., Kim A., Battist A.P., Koneripalli N., Wang Y., Chen Q., Xin R., Yang C., Huang R., Yu J., Huang Z., Zhang Z., Wang H., Wang D., Xiao Y., Salgado O.C., Jarjour N.N., Hogquist K.A., Revelo X.S., Burlingame A.L., Gao X., von Moltke J., Lin Z, & Ruan H.B. (2022). Epithelial STAT6 O-GlcNAcylation drives a concerted anti-helminth alarmin response dependent on tuft cell hyperplasia and Gasdermin C. Immunity, 55(4), 623-638.e5.
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3

Multiprobe RNA in situ Hybridization in Forebrain Organoids

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RNA in situ hybridization was performed using the RNAscope Fluorescent Multiplex Reagent Kit (Advanced Cell Diagnostics). The samples were prepared according to the manufacturer’s instructions. Briefly, forebrain organoids were collected and embedded in OCT. The fresh frozen sample were equilibrated to −20°C in a cryostat for 1 h. 15um FFPE tissue sections were mounted on SuperFrost Plus slides, fixed in 4% PFA for 15 min at 4°C, dehydrated using 50%, 70% and 100% ethanol, and treated with RNAscope protease IV (Advanced Cell Diagnostics) for 15 min at room temperature. Probes were mixed at a 50:1:1 ratio (C1: C2:C3) and hybridized for 2 h at 40°C in the HybEZ Oven (Advanced Cell Diagnostics). The following probes from Advanced Cell Diagnostics were used: Hs-SOX2 (catalog #400871-C1), Hs-DLX2-C2 (catalog #483311-C2), and Hs-PAX6-C3 (catalog #588881-C3). Amplification and detection were performed according to the manufacturer’s instructions. Slides were applied to DAPI for 30s at room temperature, added fluorescent mounting medium and covered with coverslips. All images were captured by Nikon Eclipse Ti-E microscope. Quantitative analyses were conducted randomly on 5 regions per section in a blind fashion. The area positive for each probe was measured using ImageJ software (NIH).
Kang Y., Zhou Y., Li Y., Han Y., Xu J., Niu W., Li Z., Liu S., Feng H., Huang W., Duan R., Xu T., Raj N., Zhang F., Dou J., Xu C., Wu H., Bassell G.J., Warren S.T., Allen E.G., Jin P, & Wen Z. (2021). A human forebrain organoid model of fragile X syndrome exhibits altered neurogenesis and highlights new treatment strategies. Nature neuroscience, 24(10), 1377-1391.
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4

Spinal Cord Tissue Preparation for RNAscope

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The mouse spinal cord fixed frozen tissue slide was first dehydrated by successive incubations in 50%, 70%, and 100% ethanol at room temperature, each for 5 min. Then, the slide was baked for 10 min at 60 °C. Subsequently, HRP blocking was performed via a 10 min incubation of the slide in HRP blocking buffer at room temperature, followed by 3 times 1× PBS wash at room temperature, each for 5 min. Finally, the slide was incubated with RNAscope Protease IV (Advanced Cell Diagnostics, Newark, CA, USA) for 30 min and washed with 1× PBS 3 times at room temperature, each for 5 min.
Xiao L., Labaer J, & Guo J. (2021). Highly Sensitive and Multiplexed In Situ RNA Profiling with Cleavable Fluorescent Tyramide. Cells, 10(6), 1277.
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