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Hybez oven

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The HybEZ oven is a compact, benchtop instrument designed for in-situ hybridization (ISH) applications. It provides precise temperature control and incubation capabilities to facilitate the hybridization of nucleic acid probes during ISH experiments.

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Lab products found in correlation

Rnascope multiplex fluorescent reagent kit v2, by Advanced Cell Diagnostics (4 mentions) Rnascope fluorescent multiplex reagent kit, by Advanced Cell Diagnostics (4 mentions) Rnascope protease 4, by Advanced Cell Diagnostics (3 mentions) Rnascope 2.5 hd detection reagent kit, by Advanced Cell Diagnostics (3 mentions) Sp8 confocal microscope, by Leica (3 mentions) Rnascope 2.0 hd detection kit red, by Advanced Cell Diagnostics (3 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) Eclipse ti e microscope, by Nikon (2 mentions) Rnascope 2.5 hd reagent kit brown, by Advanced Cell Diagnostics (2 mentions) Multiplex fluorescent reagent kit v2, by Advanced Cell Diagnostics (2 mentions) Rnascope multiplex fluorescent v2 kit, by Advanced Cell Diagnostics (2 mentions) Opal 570, by Akoya Biosciences (2 mentions) Rnascope probe, by Advanced Cell Diagnostics (2 mentions) Superfrost plus slide, by Thermo Fisher Scientific (2 mentions) Prolong gold antifade, by Thermo Fisher Scientific (2 mentions) Hoechst 33342, by Thermo Fisher Scientific (1 mentions) Cytoseal 60, by Electron Microscopy Sciences (1 mentions) Rnascope 2.5 hd reagent kit, by Advanced Cell Diagnostics (1 mentions)

38 protocols using hybez oven

1

Multiplexed Fluorescent In Situ Hybridization Analysis

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We utilized RNAscope® Multiplex Fluorescent Reagent Kit v2 (Advanced Cell Diagnostics, Inc.) to perform fluorescent in situ hybridization. The slides were prepared as described above. Briefly, the tissues were treated with hydrogen peroxide and protease, then hybridized with RNAscope probes for 2 hours at 40°C using the HybEZ oven (Advanced Cell D iagnostics, Inc.). The probes were then amplified and detected with tyramide signal amplification fluorescence. To detect nuclei, the slides were incubated with 1 μg/ml Hoechst 33342 (Molecular Probes). The tissue was imaged at 63X using the Leica SP8 confocal microscope. The catalog numbers of probes from Advanced Cell Diagnostics, Inc. used in this work are listed as follow: MAFB (400801-C2), RPS21 (511381-C3), OLFM3 (549051-C3), PLA2R1 (524581), COL4A3 (461861) and TNNT2 (518991-C3).
Tran T., Lindström N.O., Ransick A., Brandine G.D., Guo Q., Kim A.D., Der B., Peti-Peterdi J., Smith A.D., Thornton M., Grubbs B., McMahon J.A, & McMahon A.P. (2019). In vivo developmental trajectories of human podocyte development inform in vitro differentiation of pluripotent stem-cell derived podocytes. Developmental cell, 50(1), 102-116.e6.
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2

In-situ detection of Adamts17 mRNA

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16.5 day old mouse embryos were fixed in 4% paraformaldehyde overnight at 4 °C, processed and paraffin-embedded. Fresh 6 μm sections were used for in-situ hybridization using RNAscope (Advanced Cell Diagnostics, Newark, CA) following the manufacturer’s protocol. All steps requiring incubation at 40 °C were performed in the HybEZ Oven (Advanced Cell Diagnostics, Newark, CA). Tissue localization of the specific probe against mouse Adamts17 mRNA (#316441) was detected with the RNAscope 2.0 HD detection kit “RED”. Probes against peptidylprolyl isomerase B (Cyclophylin B, Ppib) or bacterial dihydrodipicolinate reductase (DapB) mRNA (#313911, #310043) were used as positive and negative control, respectively. Sections were counterstained with hematoxylin prior to coverslipping with Cytoseal 60 (Electron Microscopy Science, Hatfield, PA, USA).
Hubmacher D., Schneider M., Berardinelli S.J., Takeuchi H., Willard B., Reinhardt D.P., Haltiwanger R.S, & Apte S.S. (2017). Unusual life cycle and impact on microfibril assembly of ADAMTS17, a secreted metalloprotease mutated in genetic eye disease. Scientific Reports, 7, 41871.
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3

Spatial Gene Expression Analysis

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Sections (6-µm thick) were de-paraffinized and hybridized with a specific probe for mouse Fbn2 or Fbn1 mRNA (Advanced Cell Diagnostics, Hayward, CA). Hybridization and detection was performed using the RNAScope 2.0 HD Red detection kit and HybEZ™ oven (Advanced Cell Diagnostics, Hayward, CA) according to the manufacturer's instructions.
Hubmacher D., Wang L.W., Mecham R.P., Reinhardt D.P, & Apte S.S. (2015). Adamtsl2 deletion results in bronchial fibrillin microfibril accumulation and bronchial epithelial dysplasia – a novel mouse model providing insights into geleophysic dysplasia. Disease Models & Mechanisms, 8(5), 487-499.
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4

Quantitative RNA-Scope Analysis of Pkdcc in Mouse

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RNA-Scope analysis was performed on FFPE samples using a 20-base pair Pkdcc probe (#516961, Mm-Pkdcc Probe, RNAscope®, Advanced Cell Diagnostics, Newark, CA) binding to region 5961655 of Mus musculus Pkdcc (NM_134117.2). Positive [peptidylprolyl isomerase B (PpiB), pH 6, NM_011149] and negative [dihydrodipicolinate reductase (DapB), pH 6, EF191515] controls were run alongside. Sections were processed according to the RNAscope 2.5 HD Reagent Kit protocol (pH 6, PN 322350, Advanced Cell Diagnostics), with the following adjustments: 30 min target retrieval time and 30 min, 40°C, protease plus incubation. Slides were heated using a HybEZ™ Humidity Control Tray in a HybEZ™ oven (Advanced Cell Diagnostics). Sections were counterstained with Hoechst 33342 and mounted with Mowiol®. Quantification of red mRNA dots was performed using the analyze particles plug-in of Fiji (Schindelin et al., 2012 (link)). Number of of stained mRNA (red) was counted per field of vision.
Pantasis S., Friemel J., Brütsch S.M., Hu Z., Krautbauer S., Liebisch G., Dengjel J., Weber A., Werner S, & Bordoli M.R. (2022). Vertebrate lonesome kinase modulates the hepatocyte secretome to prevent perivascular liver fibrosis and inflammation. Journal of Cell Science, 135(7), jcs259243.
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5

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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6

Localization of Reproductive Hormone Receptors

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In situ hybridization (ISH) was performed using RNAscope 2.5 HD Reagent Kit-BROWN (Advanced Cell Diagnostics, Newark, CA, USA) (Wang et al. 2012 (link)) with predesigned probes for GNRHR (#407999), LHCGR (#300031), FSHR (#408101), positive control POLR2A (#310451) and nonsense dapB (from Bacillus S., #310043). Hybridization was performed according to manufacturer’s protocol in HybEZ Oven (Advanced Cell Diagnostics). Slides were scanned by Pannoramic Midi FL slide scanner (3DHISTECH Ltd.) and pictures were taken using Pannoramic Viewer (3DHISTECH Ltd.).
Doroszko M., Chrusciel M., Stelmaszewska J., Slezak T., Anisimowicz S., Plöckinger U., Quinkler M., Bonomi M., Wolczynski S., Huhtaniemi I., Toppari J, & Rahman N.A. (2018). GnRH antagonist treatment of malignant adrenocortical tumors. Endocrine-Related Cancer, 26(1), 103-117.
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7

In situ hybridization of Adamts6 and Adamts10

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Adamts6 and Adamts10 ISH was performed using RNAscope (Advanced Cell Diagnostics, Newark, CA) as described (Mead and Apte, 2020b (link)). Briefly, 6 μm sections were deparaffinized and hybridized to mouse Adamts6 and Adamts10 probe sets (428301, 585161, respectively; Advanced Cell Diagnostics) using a HybEZ oven (Advanced Cell Diagnostics) and the RNAScope 2.5 HD Detection Reagent Kit (322360; Advanced Cell Diagnostics) and counterstained with eosin.
Mead T.J., Martin D.R., Wang L.W., Cain S.A., Gulec C., Cahill E., Mauch J., Reinhardt D., Lo C., Baldock C, & Apte S.S. (2022). Proteolysis of fibrillin-2 microfibrils is essential for normal skeletal development. eLife, 11, e71142.
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8

RNAscope Assay for MeXis in Mouse Macrophages

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Custom design RNAscope probes against MeXis were prepared and obtained from Advanced Cell Diagnostics (Catalog number 495011). MeXis was visualized in immortalized mouse bone-marrow derived macrophages using an RNAscope assay and the Multiplex Fluorescent Reagent Kit V2, following the manufacturer’s recommended protocol with use of a HybEZ oven (Advanced Cell Diagnostics); however, we used a protease dilution of 1:5 instead of 1:30.
Sallam T., Jones M., Thomas B.J., Wu X., Gilliland T., Qian K., Eskin A., Casero D., Zhang Z., Sandhu J., Salisbury D., Rajbhandari P., Civelek M., Hong C., Ito A., Liu X., Daniel B., Lusis A.J., Whitelegge J., Nagy L., Castrillo A., Smale S, & Tontonoz P. (2018). Transcriptional regulation of macrophage cholesterol efflux and atherogenesis by a long noncoding RNA. Nature medicine, 24(3), 304-312.
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9

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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10

Cellular Localization of LINC00885 by FISH

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To determine the cellular localization of LINC00885, FISH was performed by utilizing the RNAscope Multiplex Fluorescent Reagent Kit v2 (Advanced Cell Diagnostics, California, USA). CC cells were subjected to 24-h incubation on the culture slides (Thermo Fisher Scientific, Inc., Waltham, MA, USA), followed by 0.5-h fixation in 4% paraformaldehyde. Then, the cell slides were treated with Hydrogen Peroxide and Protease III, followed by 2-h hybridization with the LINC00885 FISH probe (RiboBio, Guangzhou, China) in the HybEZ Oven (Advanced Cell Diagnostics). Thereafter, cells were washed and subjected to dehydration in the graded ethanol. Finally, slides were stained with DAPI (Cell Signaling Technologies, Danvers, USA). The observation and photographing of the cell slides was accomplished by using a FV1000 confocal laser microscope (Olympus, Tokyo, Japan).
Liu Y., Chen J., Zhou L, & Yin C. (2022). LINC00885 promotes cervical cancer progression through sponging miR-3150b-3p and upregulating BAZ2A. Biology Direct, 17, 4.
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