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Thermobrite system

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The ThermoBrite System is a laboratory instrument designed for the automation of in situ hybridization (ISH) and immunohistochemistry (IHC) procedures. It provides precise temperature control and incubation steps to facilitate the standardization and optimization of these molecular biology techniques.

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Lab products found in correlation

Quantigene viewrna, by Thermo Fisher Scientific (2 mentions) Lsm 700 confocal microscope, by Zeiss (2 mentions) Fish probes, by Empire Genomics (2 mentions) 80i microscope, by Nikon (2 mentions) Hoechst 33342, by Thermo Fisher Scientific (2 mentions) Dc 300 fx, by Leica camera (1 mentions) Dm 5000b fluorescence microscope, by Leica camera (1 mentions) Boehringer blocking reagent, by Roche (1 mentions) Pepsin, by Merck Group (1 mentions) 4 6 diamidino 2 phenylindole dihydrochloride dapi, by Vector Laboratories (1 mentions) Axio imager m1 microscope, by Zeiss (1 mentions) Zen software, by Zeiss (1 mentions) Plan apochromat, by Zeiss (1 mentions) Lsm780 confocal laser scanning microscope, by Zeiss (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions) Egfr fish probe, by Empire Genomics (1 mentions) Eclipse 80i fluorescent microscope, by Nikon (1 mentions) Zytolight spec cdk4 cen12 dual color probe, by ZytoVision (1 mentions) Igepal, by Merck Group (1 mentions) Leica sp5 confocal microscope, by Leica Microsystems (1 mentions)

12 protocols using thermobrite system

1

MCPyV DNA Detection by FISH

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MCPyV FISH was performed as previously described.22, 23 In brief, deparaffinized 3 μm thick sections were pretreated with 0.2 M hydrochloric acid, incubated with 1 M NaSCN and digested with 1 mg/mL pepsin (2500–3500 U/mg, Sigma Chemical, St. Louis, MO, USA). The biotin labeled “specific” MCPyV DNA probe was added to the samples at a concentration of 5 ng/μL, followed by denaturation of DNA (five minutes, 80°C) and hybridization overnight (37°C, humid chamber; ThermoBrite System, Abbott Molecular, Abbot Park, IL, USA). Unbound MCPyV DNA probe was stringently washed away. Bound probe was detected by sequential incubation in a combination of secondary antibodies: fluorescein isothiocyanate (FITC) avidin secondary antibody (1:500) and biotin conjugated goat anti‐avidin (1:100; Vector, Brunschwig Chemie, Amsterdam, The Netherlands). Prior to incubation, aspecific binding sites were blocked with Boehringer Blocking reagent (Roche,
Molecular Diagnostics Inc., South Branchburg, NJ, USA). Cell nuclei were counterstained, and cover slipped with 4,6‐diamidino‐2‐phenylindole dihydrochloride (DAPI; 0.2 μg/mL; Vectashield, Vector Laboratories, Burlingame, CA, USA). Samples were visualized using a DM 5000B fluorescence microscope (Leica, Wetzlar, Germany) coupled to an online digital camera (Leica DC 300 Fx) for independent evaluation of FISH signals by two investigators.
Chteinberg E., Klufah F., Rennspiess D., Mannheims M.F., Abdul‐Hamid M.A., Losen M., Keijzers M., De Baets M.H., Kurz A.K, & zur Hausen A. (2019). Low prevalence of Merkel cell polyomavirus in human epithelial thymic tumors. Thoracic Cancer, 10(3), 445-451.
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2

In Situ Hybridization of Mouse Retina

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Mice were transcardially perfused with ice-cold RNase-free PBS followed by 4% PFA (in RNase-free PBS). Enucleated eyes were post-fixed in RNAse-free 4% PFA, cryoprotected in 20% sucrose, and embedded in OCT and sectioned within 24 hours for in situ hybridization. QuantiGene View RNA (Affymetrix, Santa Clara, CA, USA) in situ hybridization assay was performed according to the manufacturer protocol. Briefly, 12μm cryosections were fixed overnight in 4% PFA, dehydrated through a graded series of ethanol, were subjected to 2X protease digestion for 10 minutes, postfixed with 4% PFA and hybridized with probe sets against the gene of interest for 3 hours at 40°C using a ThermoBrite system (Abbott Molecular, Des Plaines, IL, USA). Cryosections were then washed and subject to signal amplification and detection using fast red substrate, counterstained and mounted for subsequent imaging. For dual fluorescent in situ hybridization, Digoxigenin- and Fluorescein-labeled riboprobes were synthesized from full-length cDNA clones (MGC Mouse glutamine synthetase cDNA Clone Id:4224865). The hybridized mRNA was detected using the TSA-FITC/ TSA-CY5 Tyramide Signal Amplification System (PerkinElmer, Waltham, MA, USA).
Paylakhi S., Labelle-Dumais C., Tolman N.G., Sellarole M.A., Seymens Y., Saunders J., Lakosha H., deVries W.N., Orr A.C., Topilko P., John S.W, & Nair K.S. (2018). Müller glia-derived PRSS56 is required to sustain ocular axial growth and prevent refractive error. PLoS Genetics, 14(3), e1007244.
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3

In situ Hybridization of Adamts19 in Mouse Eyes

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Mice were transcardially perfused with ice-cold RNase-free PBS followed by 4% PFA (in RNase-free PBS). Eyes were enucleated post-fixed in RNAse-free 4% PFA, cryoprotected in 20% sucrose, and embedded in OCT compound and sectioned within 24 hours for in situ hybridization. QuantiGene View RNA (Affymetrix, Santa Clara, CA, USA). In situ hybridization was performed according to the manufacturer protocol. Briefly, 12μm cryosections were fixed overnight in 4% PFA, dehydrated through a graded series of ethanol, were subjected to 2X protease digestion for 10 minutes, fixed and hybridized with probe sets against Adamts19 (NM_175506 (Adamts19), TYPE1, high sensitivity with 40~50 bp DNAs) for 3 hours at 40°C using a Thermo Brite system (Abbott Molecular, Des Plaines, IL, USA). Cryosections were then washed and subject to signal amplification and detection using a Fast Red substrate, counterstained with DAPI (blue) to visualize the nuclei, and mounted for subsequent imaging. Fluorescent images were acquired using an AxioImager M1 microscope equipped with an MRm digital camera and AxioVision software, with an LSM700 confocal microscope and Zen software (Carl Zeiss Microscopy, LLC, Germany).
Koli S., Labelle-Dumais C., Zhao Y., Paylakhi S, & Nair K.S. (2021). Identification of MFRP and the secreted serine proteases PRSS56 and ADAMTS19 as part of a molecular network involved in ocular growth regulation. PLoS Genetics, 17(3), e1009458.
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4

FISH Analysis for HER2 Detection

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FISH analysis for HER2 was performed on 5 μm thick slides. Deparaffinization of sections was carried out with two 10 min immersions in bioclear, followed by three 3 min immersions in ethanol 100, 70 and 50%. Briefly, according with manufacturer protocol slides were rinsed in distilled water and immersed in pre-treatment solution at 80°C for 10 min, and in protease solution (previously warmed to 37°C) for 10 min, washed with purified water, air-dried, and dehydrated in ascending grades of alcohol. The commercially available HER2 pharmDX kit (Agilent Technologies Santa Clara, CA, USA, #K5331) was used.
Denaturation and hybridization of the tissue sections were performed using the Thermobrite system (Abbott Molecular Inc. Des Plaines, IL, USA): 75°C for 5 min for the denaturation process and 37°C for 15 hours for the hybridization of the probes. Slides were then washed with 0.4X saline- sodium citrate (SSC) solution at 70°C for 2 min and 2X SSC at room temperature for 3–5 min. Lastly, 10 μL of DAPI was applied on the slides.
Two different investigators that had no previous knowledge of the genetic, clinical and IHC results evaluated FISH analysis.
Oliveira D.M., Mirante T., Mignogna C., Scrima M., Migliozzi S., Rocco G., Franco R., Corcione F., Viglietto G., Malanga D, & Rizzuto A. (2018). Simultaneous identification of clinically relevant single nucleotide variants, copy number alterations and gene fusions in solid tumors by targeted next-generation sequencing. Oncotarget, 9(32), 22749-22768.
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5

FISH Assay for Rat Chromosomes 1 and 2

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The FISH protocol was performed according to the Metasystem guidelines. FISH assay for rat chromosomes 1 (probe XRP1 green; 405 nm), (MetaSystems, Catalog No. D-1501-FI) and 2 (probe XRP2 orange; 488 nm), (MetaSystems, Catalog No. D-1502-050-OR) was carried out at the metaphase stage, where cells were pre-treated with 10 ml of hypotonic solution (75 mM KCl)75 (link). Briefly, cells were fixed with methanol and acetic acid (3:1). Fixed cells were spotted on glass slides and spread uniformly and the slides were dried. Using the ThermoBrite system from Abbott Molecular, the sample and probe were denatured at 75 °C for 2 minutes and the temperature was then lowered to 37 °C to allow the probe to hybridize for 48 hours. The slides were counter stained with anti-fade DAPI (MetaSystems, Catalog No. CD-0902-500-DA) and covered with cover slips. Images were captured using confocal microscope (Zeiss LSM780 laser scanning confocal microscope) with 63x objective (Zeiss Plan-Apochromat) and fluorescence at absorption of 552 nm and emission of 576 nm (34 channel spectral R/FL detectors).
Chennakrishnaiah S., Tsering T., Gregory C., Tawil N., Spinelli C., Montermini L., Karatzas N., Aprikian S., Choi D., Klewes L., Mai S, & Rak J. (2020). Extracellular vesicles from genetically unstable, oncogene-driven cancer cells trigger micronuclei formation in endothelial cells. Scientific Reports, 10, 8532.
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6

In Situ Hybridization of Cochlear Neurons

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Paraffin-embedded cochlear spiral ganglion neuron tissue samples from mouse cochleae were selected for in situ hybridization with FITC-labeled lncRNA EBLN3P DNA probe (ShineGene). The paraffin-embedded tissues were cut into 5-µm-thick tissue sections for FISH using a Leica RM2235 Manual Rotary Microtome (Leica Microsystems GmbH). After dewaxing the slides with xylem (2x10 min) and methanol (2x5 min). The slides were exposed to microwaves (720 W) in 10 mM citric acid monohydrate (pH 6.0) for 30 sec, followed by immersion in 1 M sodium thiocyanate for 10 min at 80℃, and in protease solution for 10 min. The tissue sections were then washed with pure H2O, air-dried and dehydrated in ascending gradients of alcohol. The lncRNA EBLN3P probe mixtures (10 ml) were added to the air-dried tissue sections. The ThermoBrite System (Abbott Molecular) was used for denaturation and hybridization. Subsequently, the tissue sections were washed with 0.4X saline sodium citrate solution for 2 min at 70°C, and 2X saline sodium citrate solution for a further 5 min at room temperature. Finally, 10 ml DAPI (Beyotime Institute of Biotechnology) was added to the tissue sections, followed by fluorescence microscopy (Leica, Ernst-Leitz) evaluation. The final data were evaluated by two investigators.
Jiang W., Peng A., Chen Y., Pang B, & Zhang Z. (2020). Long non-coding RNA EBLN3P promotes the recovery of the function of impaired spiral ganglion neurons by competitively binding to miR-204-5p and regulating TMPRSS3 expression. International Journal of Molecular Medicine, 45(6), 1851-1863.
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7

Chromosome Sorting and Fluorescence In Situ Hybridization

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Sorted cells were spun onto slides, fixed in methanol and acetic acid, and prepared with dual-labeled XY FISH probes on a ThermoBrite System (Abbott) in accordance with the manufacturer’s instructions.
Jardine L., Cytlak U., Gunawan M., Reynolds G., Green K., Wang X.N., Pagan S., Paramitha M., Lamb C.A., Long A.K., Hurst E., Nair S., Jackson G.H., Publicover A., Bigley V., Haniffa M., Simpson A.J, & Collin M. (2020). Donor monocyte–derived macrophages promote human acute graft-versus-host disease. The Journal of Clinical Investigation, 130(9), 4574-4586.
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8

Multiprobe FISH analysis of cancer cells

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MDA-MB-231 cells were cultured until 80% confluency in a 10 cm dish and transferred to 15 ml conical tubes and centrifuged at 1500 rpm for 7 min. Cells were subjected to hypotonic treatment (0.075 M KCl) for 20 min at room temperature and fixed in methanol and acetic acid mixture (3:1 v/v) for 15 min, washed three times with the fixative and air-dried. DNA fluorescence in situ (DNA-FISH) hybridization was performed on the above cytological preparations using SHC1–20-GR, EGFR-20-GR, VEGFC-20-GR, PIK3CA-20-GR, AKT3–20-GR, FGFR3–20-GR, MET-20-OR, PDGFRA-20-OR, BCAS2–20-OR probes. (Empire Genomics, Buffalo, NY, USA). Slides were hybridized with the FISH probes according to the manufacturer’s instructions (Empire Genomics) with slight modifications. Briefly, 2 μl of each of the two probes were mixed with 6 μl of the in situ hybridization buffer. The probe was applied on the slide and covered with a glass coverslip (22X22 mm) and sealed with rubber cement. The slides were then denatured at 72–73°C using Thermobrite system (Abbott Laboratories, Illinois, USA) and incubated at 37°C overnight. The slides were then washed using 2XSSC 45–70°C for 1–2 mins, counterstained with DAPI and analyzed using Nikon 80i microscope using the green and orange fluorescent channels. The copy number states of each probe were counted across 1000 cells and multiple imaging fields for each experiment.
Minussi D.C., Nicholson M.D., Ye H., Davis A., Wang K., Baker T., Tarabichi M., Sei E., Du H., Rabbani M., Peng C., Hu M., Bai S., Lin Y.W., Schalck A., Multani A., Ma J., McDonald T.O., Casasent A., Barrera A., Chen H., Lim B., Arun B., Meric-Bernstam F., Van Loo P., Michor F, & Navin N.E. (2021). Breast Tumors Maintain a Reservoir of Subclonal Diversity During Expansion. Nature, 592(7853), 302-308.
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9

Multiprobe FISH analysis of cancer cells

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MDA-MB-231 cells were cultured until 80% confluency in a 10 cm dish and transferred to 15 ml conical tubes and centrifuged at 1500 rpm for 7 min. Cells were subjected to hypotonic treatment (0.075 M KCl) for 20 min at room temperature and fixed in methanol and acetic acid mixture (3:1 v/v) for 15 min, washed three times with the fixative and air-dried. DNA fluorescence in situ (DNA-FISH) hybridization was performed on the above cytological preparations using SHC1–20-GR, EGFR-20-GR, VEGFC-20-GR, PIK3CA-20-GR, AKT3–20-GR, FGFR3–20-GR, MET-20-OR, PDGFRA-20-OR, BCAS2–20-OR probes. (Empire Genomics, Buffalo, NY, USA). Slides were hybridized with the FISH probes according to the manufacturer’s instructions (Empire Genomics) with slight modifications. Briefly, 2 μl of each of the two probes were mixed with 6 μl of the in situ hybridization buffer. The probe was applied on the slide and covered with a glass coverslip (22X22 mm) and sealed with rubber cement. The slides were then denatured at 72–73°C using Thermobrite system (Abbott Laboratories, Illinois, USA) and incubated at 37°C overnight. The slides were then washed using 2XSSC 45–70°C for 1–2 mins, counterstained with DAPI and analyzed using Nikon 80i microscope using the green and orange fluorescent channels. The copy number states of each probe were counted across 1000 cells and multiple imaging fields for each experiment.
Minussi D.C., Nicholson M.D., Ye H., Davis A., Wang K., Baker T., Tarabichi M., Sei E., Du H., Rabbani M., Peng C., Hu M., Bai S., Lin Y.W., Schalck A., Multani A., Ma J., McDonald T.O., Casasent A., Barrera A., Chen H., Lim B., Arun B., Meric-Bernstam F., Van Loo P., Michor F, & Navin N.E. (2021). Breast Tumors Maintain a Reservoir of Subclonal Diversity During Expansion. Nature, 592(7853), 302-308.
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10

EGFR-FISH Assay for Glioblastoma

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A fluorescence in situ hybridization (FISH) assay was performed on human and mouse glioblastoma tumor slides using an EGFR-FISH probe from Empire Genomics, as per the manufacturer’s instructions, as described previously.29 (link) In brief, the probe was applied to the slides, covered with a glass coverslip, and sealed with rubber cement. The slides were denatured at 70°C using the ThermoBrite system (Abbott Laboratories, Chicago, IL, USA) and incubated at 37°C overnight. The slides were washed with sodium citrate buffer at 45°C for 1–2 min, rinsed in PBS containing 0.05% v/v Tween-20, and counterstained with DAPI. The slides were analyzed under a Nikon ECLIPSE 80i fluorescent microscope. Randomly chosen fields of at least 50 cells were quantified for EGFR and centromere 7 (CEP7) amplification. The FISH data were analyzed by manually counting cells with EGFR or CEP7 staining. An EGFR/CEP7 ratio of >2 was considered focal amplification, whereas a ratio of <2 was considered broad amplification.
Khan S., Martinez-Ledesma E., Dong J., Mahalingam R., Park S.Y., Piao Y., Koul D., Balasubramaniyan V., de Groot J.F, & Yung W.K. (2023). Neuronal differentiation drives the antitumor activity of mitogen-activated protein kinase kinase (MEK) inhibition in glioblastoma. Neuro-Oncology Advances, 5(1), vdad132.
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