Lsi wcp hybridization buffer

Manufactured by Abbott

Sourced in United States

The LSI/WCP hybridization buffer is a laboratory reagent used in the hybridization process during molecular biology and genetics experiments. It is designed to facilitate the binding of labeled DNA or RNA probes to their target sequences on membranes or slides.

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Lab products found in correlation

4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (3 mentions) Nick translation kit, by Abbott (2 mentions) Lsm 700 confocal microscope, by Zeiss (2 mentions) Vysis cep y, by Abbott (1 mentions) Prolong gold, by Thermo Fisher Scientific (1 mentions) Green dutp, by Abbott (1 mentions) Dna clean concentrator 5, by Zymo Research (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions) Dxz1 dyz3, by Abbott (1 mentions) Cytovision, by Leica (1 mentions) Thermobrite, by Abbott (1 mentions)

8 protocols using lsi wcp hybridization buffer

FFPE Tissue FISH Probe Detection

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FFPE tissue slides were de-paraffinized and washed with xylene and EtOH. Slides were pretreated, hybridized with FISH probes, and then mounted for microscope observation. Pretreatment was performed using Pretreatment Kit I (02J02-032, Abbott Molecular). Probes were mixed with LSI/WCP Hybridization Buffer (06J67-001, Abbott Molecular). Slides were counterstained with DAPI, and mounted with ProLong® Gold (P36930, Invitrogen). All fluorescence microscopy photos were imaged with NIS-Elements BR2.30. All FISH probes used were from Abbott Molecular: Vysis CEP X (DXZ1) SO Probe (centromeric, 05J08-033), Vysis CEP X (DXZ1) SA Probe (centromeric, 05J09-033), Vysis CEP Y (DYZ1) SGn Probe (q arm, 05J10-034), Vysis CEP Y (DYZ3) SO Probe (centromeric, 05J08-035), and Vysis LSI SRY SO Probe (p arm, 05J27-089).

Wong H.Y., Wang G.M., Croessmann S., Zabransky D.J., Chu D., Garay J.P., Cidado J., Cochran R.L., Beaver J.A., Aggarwal A., Liu M.L., Argani P., Meeker A., Hurley P.J., Lauring J, & Park B.H. (2015). TMSB4Y is a candidate tumor suppressor on the Y chromosome and is deleted in male breast cancer. Oncotarget, 6(42), 44927-44940.

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BAC Probe Labeling with Green dUTP

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The BAC probe labeling and preparation was adapted from [98 (link)]. The probe was labelled with Green dUTP (Abbott Molecular, Abbott Park, IL, Catalog# 02N32-050) using the Nick Translation Kit (Abbott Molecular, Catalog# 07J00-001). 14 μl of the purified BAC was mixed with 23.4 μl of 0.1 mM dNTP mix (1:2:2:2 of dTTP:dATP:dCTP:dGTP), 10 μl of 10X Nick translation buffer, 10 μl of the Nick translation enzyme mix, 12 μl of 0.2 mM Green dUTP, and H₂O to bring up the volume to 100 μl. The reaction was incubated in a thermocycler at 15°C for 16 hours, heated to 70°C for 10 minutes, and then held at 4°C. The labeled BAC was purified using DNA Clean & Concentrator-5 (Zymo Research, Irvine, CA, Catalog# D4013) in 50 μl batches and eluted in 10 μl of the elution buffer. 25 μg of salmon sperm Cot-1 was added per batch and the batches were mixed together. The mixture was vacuum dried, and the pellet was resuspended in 10 μl of LSI buffer (LSI/WCP Hybridization Buffer, Abbott Molecular, Catalog# 06J67-011) to make the stock BAC probe mix. The stock was stored in the dark at -20°C. For staining, the stock was further diluted in LSI buffer at a 1:19 stock:LSI ratio.

Blokhina Y.P., Nguyen A.D., Draper B.W, & Burgess S.M. (2019). The telomere bouquet is a hub where meiotic double-strand breaks, synapsis, and stable homolog juxtaposition are coordinated in the zebrafish, Danio rerio. PLoS Genetics, 15(1), e1007730.

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HSET Gene Copy Number Analysis

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The samples from tumor cell lines or tumor tissue were hybridized by 2-color FISH with an HSET-specific BAC probe (RPCI-11 602P21, green) and a chromosome 6 centromere probe (CH514-7B4, red) (BACPAC). The HSET and centromere 6 probes were labeled with Cy3-dUTP (red) and FITC-dUTP (green), respectively, and hybridized with nuclei from cell lines or tumor tissue samples. Plasmids for production of a particular FISH probe were combined in equimolar amounts (55–70 pM). Nick translation was performed on 2 μg of this substrate by using Nick translation kit (Abbott Molecular, IL, USA). The translation product was denatured for 3 mins at 95°C followed by fast cooling on ice and confirmed in 1.5% agarose gel electrophoresis as a smear of fragments ranging between 100 and 300 bp. A 2 min denaturation at 76°C was followed by overnight (12–16h) incubation at 37°C. Hybridization of the FISH probes was carried out in LSI/WCP hybridization buffer (Abbott Molecular, IL, USA). The slides were counterstained with DAPI (Invitrogen, NY, USA) and Zeiss LSM 700 confocal microscope was used to capture FISH images. Results were expressed as a ratio of the number of copies of the HSET gene to the number of chromosome 6–centromeric markers.

Pannu V., Rida P.C., Ogden A., Turaga R.C., Donthamsetty S., Bowen N.J., Rudd K., Gupta M.V., Reid M.D., Cantuaria G., Walczak C.E, & Aneja R. (2015). HSET overexpression fuels tumor progression via centrosome clustering-independent mechanisms in breast cancer patients. Oncotarget, 6(8), 6076-6091.

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FISH Detection of i(12p) in Tumors

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FISH was performed on unstained slides prepared from archival tissue at the Mayo Clinic Cytogenetics Core to detect the presence of i(12p). FISH was also performed on 1 test case at the MDACC Cytogenetics Laboratory for confirmation of the results and with matched normal tissue for a negative control and the detection of germline i(12p). In brief, the working solution contained 3 μL concentrated PKP2 DNA probe labeled with SpectrumOrange and 1 μL of concentrated D12Z3 probe labeled with SpectrumGreen (Abbott Molecular/Vysis Products, Abbott Park, IL, USA) in 16 μL of LSI/WCP hybridization buffer (Abbott Laboratories). Two technologists independently scored 50 qualifying tumor nuclei for each sample. In the event of a discrepancy, a third technologist would also score the sample and report the original score closest to its value. The normal cutoff value for enumeration of the i(12p) probe set with 95% (p < 0.05) confidence level was 30% [43 (link)].

Umbreit E.C., Siddiqui B.A., Hwang M.J., Joon A.Y., Maity T., Westerman M.E., Merriman K.W., Alhasson H., Uthup J., Guo T., Moore J.A., Ward J.F., Karam J.A., Wood C.G., Pisters L.L., Zhang M, & Tu S.M. (2020). Origin of Subsequent Malignant Neoplasms in Patients with History of Testicular Germ Cell Tumor. Cancers, 12(12), 3755.

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Chromosome 8 Painting Probe Hybridization

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Probes were labeled using the Nick Translation Kit (Abbot Molecular/Vysis, Des Plaines, IL, USA). Cells were fixed, washed, and dried on slides. 10 μL of chromosome 8 painting probe (Cytocell, Oxford Gene Technology, Tarrytown, NY, USA) were mixed with 1–2 μL of labeled probe and hybridized for 2 min at 72 °C in LSI/WCP hybridization buffer (Abbott Molecular/Vysis) followed by overnight at 37 °C. The following day, slides were washed and mounted with 10 μL of DAPI with antifade (Cytocell). For details, see Supplemental materials and methods.

Gottschalk L.B., Vecchio-Pagan B., Sharma N., Han S.T., Franca A., Wohler E.S., Batista D.A., Goff L.A, & Cutting G.R. (2015). Creation and characterization of an airway epithelial cell line for stable expression of CFTR variants. Journal of cystic fibrosis : official journal of the European Cystic Fibrosis Society, 15(3), 285-294.

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Fetal Sex Identification by FISH

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Six placental samples from a male fetus were used to identify infiltrating cell origin by fluorescence in situ hybridization (FISH). Slides were placed in a 90°C oven for 15 minutes. Slides were then deparaffinized with xylene (2 times, 15 minutes each) at room temperature (RT), dehydrated in 100% ethanol for 5 minutes at RT, and placed in 10mM Citric Acid (pH 6.0) and microwaved for 10 minutes. The slides were then dehydrated using an ethanol series (70, 85, 100%) 2 minutes each at RT. Working solution of DXZ1/DYZ3 (Abbott Laboratories, Des Plaines, IL, USA) was made by mixing 1 µL of concentrated DXZ1/DYZ3 probe with 9 µL of LSI/WCP hybridization buffer (Abbott Laboratories). The working solution was applied to the target areas, cover slipped, co-denatured with a ThermoBrite (Abbott Laboratories) at 83°C for 5 minutes, and hybridized overnight in a 37°C humidified oven. Following hybridization, slides were soaked in RT 2xSSC/0.1% NP-40 to remove coverslips, placed in 2xSSC/0.1% NP-40 at 74°C for 2 minutes and then placed into RT 2xSSC/0.1% NP-40 for 2 min. The slides were stained with 4’−6,-diamidino-2-phenylindole (DAPI) (Vector Laboratories) and cover slipped. Tissue samples were scanned in their entirety and the qualitative result was determined based on observed signal patterns by CytoVision (Leica Biosystems, Wetzlar, Germany).

Enninga E.A., Raber P., Quinton R.A., Ruano R., Ikumi N., Gray C.M., Johnson E.L., Chakraborty R, & Kerr S.E. (2020). Maternal T cells in the Human Placental Villi support an Allograft Response during Non-Infectious Villitis. Journal of immunology (Baltimore, Md. : 1950), 204(11), 2931-2939.

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Fluorescence In Situ Hybridization Protocol

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Slides were placed in a 90°C oven for 15 minutes. Slides were then deparaffinized with xylene (2 times, 15 minutes each) at room temperature (RT), dehydrated in 100% ethanol for 5 minutes at RT, and placed in 10mM Citric Acid (pH 6.0) and microwaved for 10 minutes. Following this, the slides were immersed in 2x standard saline citrate (SSC) for 5 minutes at 37°C followed by digestion in 0.2 % pepsin working solution (1.2 grams pepsin/600 mL 0.9% NaCl pH 1.5) at 37°C for 12 minutes. Immediately after digestion, the slides were dehydrated using an ethanol series (70, 85, 100%) 2 minutes each at RT. Working solution of GATA4/3’FGFR2 (Mayo Clinic laboratory developed probe) was made by mixing 2 °L of concentrated 3’FGFR2 probe and 1 μL of concentrated GATA4 probe with 7 μL of LSI/WCP^® hybridization buffer (Abbott Laboratories). The working solution was applied to the target areas, coverslipped, co-denatured with a ThermoBrite^® at 83°C for 5 minutes, and hybridized overnight in a 37°C humidified oven. Following hybridization, slides were soaked in RT 2xSSC/0.1% NP-40 to remove coverslips, placed in 2xSSC/0.1% NP-40 at 74°C for 2 minutes and then placed into RT 2xSSC/0.1% NP-40 for 2 min. The slides were stained with 4’−6,-diamidino-2-phenylindole (DAPI) (Vector Laboratories) and coverslipped.

Dubois F.P., Shapira O., Greenwald N.F., Zack T., Wala J., Tsai J.W., Crane A., Baguette A., Hadjadj D., Harutyunyan A.S., Kumar K.H., Blattner-Johnson M., Vogelzang J., Sousa C., Kang K.S., Sinai C., Wang D.K., Khadka P., Lewis K., Nguyen L., Malkin H., Ho P., O’Rourke R., Zhang S., Gold R., Deng D., Serrano J., Snuderl M., Jones C., Wright K.D., Chi S.N., Grill J., Kleinman C.L., Goumnerova L.C., Jabado N., Jones D.T., Kieran M.W., Ligon K.L., Beroukhim R, & Bandopadhayay P. (2022). Structural variants shape driver combinations and outcomes in pediatric high-grade glioma. Nature cancer, 3(8), 994-1011.

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FISH-based Identification of ID2 and MYCN

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Slides were air dried at RT overnight. Following this, the slides were immersed in 2x standard saline citrate (SSC) for 30 minutes at 37°C. The slides were dehydrated using an ethanol series (70, 85, 100%) 2 minutes each at RT. Working solution of ID2/MYCN was made by mixing 2 μL of concentrated ID2 probe and 1 μL of concentrated MYCN probe with 7 μL of LSI/WCP^® hybridization buffer (Abbott Laboratories). The working solution was applied to the target areas, coverslipped, co-denatured with a ThermoBrite^™ at 73°C for 5 minutes and hybridized overnight in a 37°C humidified oven. Following hybridization, slides were soaked in RT 2xSSC/0.1% NP-40 to remove coverslips, placed in 2xSSC/0.1% NP-40 at 74°C for 2 minutes and then placed into RT 2xSSC/0.1% NP-40 for 2 min. The slides were stained with 10% 4’−6,-diamidino-2-phenylindole (DAPI) (Vector Laboratories) and coverslipped.

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