Hybrite

Manufactured by Abbott

Sourced in United Kingdom

HYBrite is a laboratory instrument designed for performing various hybridization processes. It offers precise temperature control and agitation capabilities to facilitate efficient and consistent hybridization reactions.

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Lab products found in correlation

Cytovision 7.2 spot counting system, by Leica (3 mentions) 4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (3 mentions) Bx61 fluorescence microscope, by Olympus (3 mentions) Vysis lsi spectrumorange c myc probe, by Abbott (2 mentions) Cep8 spectrumgreen probe, by Abbott (2 mentions) Igh myc cep8 tri colour dual fusion translocation probe, by Abbott (2 mentions) Axioscop 2 microscope, by Zeiss (1 mentions) Pathvysion her2 dna probe kit, by Abbott (1 mentions) Pepsin solution, by Agilent Technologies (1 mentions) Anti dig hrp, by Roche (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Roche (1 mentions) Dm2500, by Leica (1 mentions) Nick translation kit, by Abbott (1 mentions) Pretreatment solution, by Abbott (1 mentions) Protease solution, by Abbott (1 mentions) Pathvysion assay, by Abbott (1 mentions) Igh break apart, by Abbott (1 mentions) Axioscope 40, by Zeiss (1 mentions) 4 6 diamidino 2 phenylindole dapi, by Oxford Gene Technology (1 mentions) Bx50 microscope, by Olympus (1 mentions)

15 protocols using hybrite

Evaluation of Breast Cancer Biomarkers

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HER2, ER and PGR positivity of cases of the FIRB cohort were re-evaluated (HER2: A0485; ER: clone 1D5, M7047; PGR: clone PgR636, M3569, Dako, Santa Clara, CA, USA) on formalin-fixed, paraffin-embedded (FFPE) tissue sections. The immunoreaction was developed using the streptavidin–biotin–peroxidase technique, followed by counterstaining with Carazzi hematoxylin. HER2 positivity was defined as 3+ overexpression in more than 10% of tumor cells by IHC or 2+ overexpression and HER2 amplification ratio of at least 2.2 by FISH. Tumors were considered ER- or PGR-positive if at least 10% of cells showed immunoreactivity.
All HER2 2+ cases were evaluated by FISH using the PathVysion HER-2DNA Probe kit (Abbott, Chicago, IL, USA) according to the manufacturer’s recommendations, as previously described [15 (link)]. Briefly, 2 µm FFPE sections were deparaffined in Hybrite (Abbott) and rehydrated. After pepsin treatment (0.01 N HCl + 0.4% pepsin) at 37 °C for 6 min, samples were denatured at 85 °C for 1 min and hybridized with probes overnight at 37 °C. Samples were stained with 4′,6-diamidino-2-phenylindole, cover-slipped and analyzed with a Zeiss Axioscop 2 microscope (Carl Zeiss, Oberkochen, Germany).

Rybinska I., Sandri M., Bianchi F., Orlandi R., De Cecco L., Gasparini P., Campiglio M., Paolini B., Sfondrini L., Tagliabue E, & Triulzi T. (2020). Extracellular Matrix Features Discriminate Aggressive HER2-Positive Breast Cancer Patients Who Benefit from Trastuzumab Treatment. Cells, 9(2), 434.

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FISH Probes for MIR31 and CDKN2A Loci

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FISH probes for the MIR31 locus (chromosome 9p21) and the CDKN2A locus (chromosome 9p22) were prepared by labeling BAC-PAC clones RP11-354P17 and RP11-149I2, respectively, (Children’s Hospital and Research Center). DNA was labeled by nick translation (Abbott Laboratories, Des Plaines, IL) using Spectrum Green dUDP for RP11-354P17 (MIR31) and Spectrum Orange dUDP for RP11-149I2 (CDKN2A) following manufacturer’s protocol. Paraffin tissue sections were processed according to standard protocols, including a protease step. Probe hybridization was performed in a HYBrite (Abbott) with denaturation at 73°C for 6 min and overnight hybridization at 37°C. The slides were washed, DAPI counterstained, and viewed by fluorescence microscopy with Applied Imaging CytoVision software.

Thompson M.A., Edmonds M.D., Liang S., McClintock-Treep S., Wang X., Li S, & Eischen C.M. (2015). miR-31 and miR-17-5p levels change during transformation of follicular lymphoma. Human pathology, 50, 118-126.

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miR-214 FISH Localization in NPC

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Fluorescence in situ hybridization (FISH) of miR-214 was performed on 5 µm tissue sections of NPC following the manufacturer's instructions. Briefly, sections were at 59°C 2 h to attach cores to the silane-coated slide. Then, sections were de-paraffinised with xylene two times for 5 minutes each, rehydrated with ethanol (100 - 50 - 25% for 5 min each), and treated with DEPC water for 1 min. Subsequently, sections were treated with pepsin solution (1.3 mg/ml) (Dako, Glostrup, Denmark) at 37°C for 30 min. Following a post-fixation step in 4% paraformaldehyde (PFA), LNA-miR-214 detection probe (Exiqon, Vedbek, Denmark) or LNA-control (Exiqon, Vedbek, Denmark) was hybridized to the sections at 58°C for 4 hours carried out in a Hybrite (Abbott Laboratories, Shanghai, China). After post-hybridization wash, sections were incubated with anti-DIG-HRP (Roche, Shanghai, China) in a heater at 37°C for 30 min. Slides were washed by TNT buffer for 15 min at room temperature (RT) and incubated with Fluorophore TSA Reagent working solution for 10 min at RT. The slides were counterstained with 4′, 6-diamidino-2-phenylindole (DAPI) (Roche Diagnostics, Shanghai, China) and visualized the nuclei and glass mounting by fluorescence microscopy with a Jenoptik camera and VideoTesT-FISH 2.0 software (GP Medical Technologies Ltd, Beijing, China).

Zhang Z.C., Li Y.Y., Wang H.Y., Fu S., Wang X.P., Zeng M.S., Zeng Y.X, & Shao J.Y. (2014). Knockdown of miR-214 Promotes Apoptosis and Inhibits Cell Proliferation in Nasopharyngeal Carcinoma. PLoS ONE, 9(1), e86149.

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Cytogenetic Analysis of Chromosome Translocations

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Peripheral blood was collected and metaphase chromosome preparations were obtained from phytohemagglutinin-stimulated lymphocyte cultures according to standard procedures (Silva et al, 2018) . Routine cytogenetic analysis by G-banding techniques at the 550 bands of resolution was performed. Metaphases were examined under a Leica DM2500 (Leica, Wetzlar, Germany) microscope using 20X and 100X objective lenses. Locus-specific florescence in situ hybridization (FISH) was performed to verify translocation breakpoints. The FISH probes were set up using bacterial artificial SpectrumGreen-dUTP or SpectrumOrange-dUTP by using the Nick Translation kit (Abbott Laboratories, Abbott Park, IL) according to the manufacturer's protocol. FISH analysis was performed on chromosome spread using probes: RP11-633A13 (green), N0693H11 (orange) and RP11-727K24
(orange) specific for 17q24.3 region while RP11-706N07 (green) and RP11-147B11 (green) specific for 1p32.1 and 1p32.2 regions, respectively. FISH was performed by co-denaturing probes and chromosomes at 74 °C for 3 min, and keeping the slides into the hybridisation chamber (Hybrite; Abbott Laboratories, Abbott Park, IL) at 37 °C overnight. After three washes, metaphases were counterstained with 4',6-diamidin-2-fenilindol (DAPI) (Leica, Wetzlar, Germany).

Cinque L., Micale L., Manara E., Esposito A., Palumbo O., Chiariello A.M., Bianco S., Guerri G., Bertelli M., Giuffrida M.G., Bernardini L., Notarangelo A., Nicodemi M, & Castori M. (2022). A novel complex genomic rearrangement affecting the KCNJ2 regulatory region causes a variant of Cooks syndrome. Human genetics, 141(2).

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FISH Assay for 1p/19q Status

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FISH with Vysis probes was used to assess 1p/19q status. Sections (3 μm thick) were first deparaffinized in xylene, incubated with 0.3% pepsin in 10 mM HCl at 37℃ for 10 minutes, and boiled with citrate buffer (pH 6.0) in a microwave. Sections were then incubated in 1 M NaSCN for 35 minutes at 80℃, immersed in the pepsin solution, and fixed in 10% neutral buffered formalin. 1p36/1q25 and 19q13/19p13 labeled locusspecific dual-color probes (Abbott Molecular) were used according to the manufacturer’s protocol. We applied the probe mixture to the slides and incubated them in a humidified atmosphere with HYBrite (Abbott Molecular) at 73℃ for 5 minutes for simultaneous denaturation of the probe and target DNA. Then, we cooled the samples to 37℃ and incubated for 19 hours to hybridize the probe and target DNA. The slides were submerged in 0.4× SSC buffer/0.3% NP-40 for 2 minutes at room temperature, followed by incubation in 2× SSC/0.1% NP-40 for 5 minutes at 73℃.

Kim S.I., Lee Y., Won J.K., Park C.K., Choi S.H, & Park S.H. (2017). Reclassification of Mixed Oligoastrocytic Tumors Using a Genetically Integrated Diagnostic Approach. Journal of Pathology and Translational Medicine, 52(1), 28-36.

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Fluorescence In-Situ Hybridization for HER2 and CIN

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To identify the HER2 status and CEP17 copy number in each case, HER2 FISH (PathVysion assay, Abbott Molecular, Downers Grove, IL) was performed on TMAs of the first set and all tissue sections of the second set. FISH using CEP1 [Vysis CEP1 (D1Z5) SpectrumOrange Probe, Abbott Molecular], CEP8 [Vysis CEP8 (D8Z2) SpectrumGreen Probe, Abbott Molecular], CEP11 [Vysis CEP11 (D11Z1) SpectrumGreen Probe, Abbott Molecular], and CEP16 probe [Vysis CEP16 (D16Z3) SpectrumGreen Probe, Abbott Molecular] was performed on TMAs to assess CIN. These CEP probes around the centromere have been reported to show frequent copy number gains in breast cancer^{17 (link),23 (link),28 (link)}.
Briefly, 4 μm deparaffinized tissue sections were incubated in pretreatment solution (Abbott Molecular) at 80 °C for 30 min and then, in protease solution (Abbott Molecular) at 37 °C for 20 min. Probes were diluted in tDen-Hyb-2 hybridization buffer (Insitus Biotechnologies, Albuquerque, NM). The probes and the DNA from the tissue sections were denatured together by incubating them for 5 min at 73 °C in HYBrite™ (Abbott Molecular), and then hybridized for 16 h at 37 °C. Post-hybridization washes were performed according to the manufacturer’s protocol. The mounted slides were viewed using a fluorescence microscope.

Lee K., Kim H.J., Jang M.H., Lee S., Ahn S, & Park S.Y. (2019). Centromere 17 copy number gain reflects chromosomal instability in breast cancer. Scientific Reports, 9, 17968.

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Fluorescent In Situ Hybridization Assay for c-MYC and Centromere 8 in MCF-10A and HuMECs

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c-MYC and centromere 8 copy number status of MCF-10A cell populations was determined by dual hybridisation with a Vysis LSI SpectrumOrange c-MYC Probe (Abbott Molecular, Maidenhead, UK; cat. no.: 05J545-011) and a CEP8 SpectrumGreen Probe (Abbott Molecular; cat. no.: 06J37-018) as recommended by the suppliers. c-MYC (orange), centromere 8 (aqua) and IGH (green) copy number status of HuMECs was determined using the Abbott IGH/MYC/CEP8 Tri-colour Dual Fusion Translocation Probe (Abbott Molecular). Slides were heated to 72 °C for 5 min and then incubated for 24 h at 37 °C in a humidified hybridisation chamber (HYBrite; Abbott Molecular). After hybridisation, slides were counterstained with 4′,6-diamidino-2-phenylindole (Vector Laboratories, Peterborough, UK). FISH was scored with an Olympus BX-61 fluorescence microscope (Olympus, Southend-on-Sea, UK) with a ×100 oil objective. Images were analysed using the CytoVision 7.2 SPOT counting system (Leica Microsystems, Gateshead, UK). A minimum of 100 (for MCF-10A) or 70 (for HuMECs) nuclei were scored per test by two independent analysts.

Wade M.A., Sunter N.J., Fordham S.E., Long A., Masic D., Russell L.J., Harrison C.J., Rand V., Elstob C., Bown N., Rowe D., Lowe C., Cuthbert G., Bennett S., Crosier S., Bacon C.M., Onel K., Scott K., Scott D., Travis L.B., May F.E, & Allan J.M. (2014). c-MYC is a radiosensitive locus in human breast cells. Oncogene, 34(38), 4985-4994.

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Identification of CCND1 Translocations by FISH

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Fluorescent in-situ Hybridization (FISH) was performed with IGH/CCND1 XT, IGH break apart (Abbott Molecular, Downers Grove, IL) and MTCP1 (Empire Genomics, Williamsville, NY) probes. FISH was done according to the manufacturer’s recommendations, except prior to hybridization slides were pretreated with pepsin and postfix solution. Co-denaturation of probe and sample was done on HyBrite (Abbott Molecular, Downers Grove, IL) for 5 min at 73 C. Hybridization was carried out overnight at 37 C, and slides were washed in 0.4 x SSC/0.3%NP-40 for 2 min at 73 C. The signals were viewed using a fluorescent microscope (Zeiss Axioscope 40) equipped with appropriate filters and analyzed with Applied Imaging System.

Walker J.S., Hing Z.A., Sher S., Cronin J., Williams K., Harrington B., Skinner J.N., Cempre C.B., Gregory C.T., Pan A., Yano M., Beaver L.P., Walker B.R., Labanowska J.M., Heerema N.A., Mrózek K., Woyach J.A., Ruppert A.S., Lehman A., Ozer H.G., Coppola V., Yan P., Byrd J.C., Blachly J.S, & Lapalombella R. (2021). Rare t(X;14)(q28;q32) translocation reveals link between MTCP1 and chronic lymphocytic leukemia. Nature Communications, 12, 6338.

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Fluorescent In Situ Hybridization of Splenic Cells

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Several splenic regions were touched onto slides coated with poly-L-lysine, fixed and air-dried. The following FISH probes were applied: CEP10, CEP12 (Cytocell, Cambridge, Cambridgeshire, UK) and 11q (Abbott Molecular, Maidenhead, UK). Hybridisations were performed with 2 min denaturation at 73°C and 16 h incubation at 37°C on a HyBrite (Abbott Molecular) and subsequent application of 4′6-diamidino-2-phenylindole (DAPI) (Cytocell, Cambridge, Cambridgeshire, UK). Cells (>200/sample) were visualised on an Olympus BX50 microscope (Southend-on-Sea, Essex, UK).

Oldreive C.E., Skowronska A., Davies N.J., Parry H., Agathanggelou A., Krysov S., Packham G., Rudzki Z., Cronin L., Vrzalikova K., Murray P., Odintsova E., Pratt G., Taylor A.M., Moss P, & Stankovic T. (2015). T-cell number and subtype influence the disease course of primary chronic lymphocytic leukaemia xenografts in alymphoid mice. Disease Models & Mechanisms, 8(11), 1401-1412.

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FISH Validation for ALK Rearrangement Detection

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FISH studies were performed using ALK break apart probe [3′-spectrum orange, 5′-spectrum green] (Empire Genomics, Buffalo, NY, USA) and chromosome 2 centromere enumeration probe (CEP2) [spectrum aqua] (Abbott Molecular, Des Plaines, IL, USA) in all cases. Three micron thick tissue sections were de-paraffinized and pretreated using a Dako (Dako, Carpinteria, CA, USA) de-paraffinization and pretreatment technique followed by hybridization with a probe mixture (ALK and CEP2) of 10 µl at 45 °C on Hybrite (Abbott Molecular). After hybridization, the slides were washed and counterstained with DAPI.
ALK probe validation was carried out on FFPE sections from non-tumor breast tissue and tonsil tissue. A total of 5000 cells were evaluated and scored along with the CEP2 [spectrum aqua]. Ninety-seven percent of non-tumor cells had 2 aqua and 2 ALK fusion signals (yellow), the other 3 % showed variations of normal signal patterns (Fig. 2). The reference range was determined to be <6 % of cells, whereas any abnormal signal pattern in ≥6 % of cells was considered abnormal.Fig. 2

FISH of normal breast terminal duct lobule showing normal disomy for ALK [3′-spectrum orange, 5′-spectrum green] signals (yellow); a normal pattern in non-tumor breast cells. Inset CEP2 probe [spectrum aqua] performed on metaphase and control cells with normal 2 signal pattern

Hanna M.G., Najfeld V., Irie H.Y., Tripodi J, & Nayak A. (2015). Analysis of ALK gene in 133 patients with breast cancer revealed polysomy of chromosome 2 and no ALK amplification. SpringerPlus, 4, 439.

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