FISH was used to identify genomic TP53 deletions and translocations. A dual color FISH probe was constructed from a spectrum green labeled BAC clone (BACs RP11-89D11, RP11-404G1; Source Bioscience, Nottingham, UK) and a commercial spectrum orange labeled centromere 17 (CEP17) reference probe (no. 06J36-06; Abbott Molecular, Wiesbaden, Germany). Freshly cut 4 µm TMA sections were used for dual color FISH. Before hybridization, sections were deparaffinized and proteolytically pretreated with a commercial kit (paraffin pretreatment reagent kit; Abbott Molecular), followed by dehydration in 70, 80 and 96% ethanol, air drying and denaturation for 10 min at 72°C in 70% formamide-23 saline-sodium citrate (SSC) solution. Hybridization was done overnight at 37°C in a humidified chamber, slides were washed and counterstained with 0.2 mmol/l 40-6-diamidino-2-phenylindole in an antifade solution.
Paraffin pretreatment reagent kit
Manufactured by Abbott
Sourced in Germany
The Paraffin Pretreatment Reagent Kit is a laboratory equipment product designed for the pretreatment of paraffin-embedded tissue samples. It contains a set of reagents necessary for the deparaffinization and rehydration of tissue sections prior to further analysis or processing.
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4 protocols using paraffin pretreatment reagent kit
1
TP53 Genomic Alterations by FISH
2
FISH Analysis of IPMN Chromosomes
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Fluorescence In Situ Hybridization (FISH) was performed on paraffin-embedded IPMN tissue pretreated using the Paraffin Pretreatment Reagent Kit (Abbott Molecular Inc., Germany) following the manufacturer's protocol. Two Abbott Molecular's Vysis probes (Abbott Molecular Inc., Germany) for chromosome 3 and for MYC gene and chromosome 8 were employed according to manufacturer's instructions. Specifically, the Vysis CEP 3 (D3Z1) SpectrumOrange Probe was used to probe the 3p11.1-q11.1 Alpha Satellite DNA (orange), while the IGH/MYC/CEP 8 probe identified the IGH gene on chr14q32 (green), the MYC gene on chr8q24 (orange) and the centromeric 8p11.1-q11.1 (aqua). Haematoxylin-eosin (H&E) stained sections (4 mm) were cut before FISH sections (4 mm), to confirm tumor presence.
3
FISH Analysis of FGFR2 Amplification in iCCA
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FISH analysis was performed on formalin-fixed, paraffin-embedded (FFPE) iCCA tissue samples pre-treated with the Paraffin Pretreatment Reagent Kit (Abbott Molecular Inc., Germany), following the manufacturer’s protocol. Two Empire Genomics probes (Empire Genomics, Buffalo, NY) were employed following the manufacturer’s protocol. Specifically, the FGFR2 gene probe (orange) maps on Chromosome 10q26.13 and the CEP10 probe maps to the centromeric region of Chromosome 10. Tumour cells were counterstained with 40,6-diamidino-2-phenylindole (DAPI) for nuclear detection. Analysis was performed using an Olympus BX53 microscopy equipped with the appropriate filter sets and CytoVision software (Leica Biosystems, Nussloch, Germany). Ratio of FGFR2 signals to CEP10 signals (FGFR2/CEP10 ratio) > 2 was interpreted as gene amplification, as previously reported26 (link).
4
FISH for Urothelial Carcinoma Evaluation
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For FISH, 5-μm paraffin sections were placed in xylene for 30 (3 × 10) minutes followed by dehydration in 100% ethanol for 10 (2 × 5) minutes. Prior to hybridization, the slides were treated with Paraffin Pretreatment Reagent Kit (Abbott Molecular Inc., Des Plaines, IL, USA, Cat. # 32–801,200) and dehydrated in 70%, 85%, and 100% alcohol solutions for 5 min each. FISH was performed using UroVysion probe (Abbott Molecular, Des Plaines, IL). Denaturation of the DNA was carried out at 75 °C for 10 min (probe mixture) or 5 min (slides). The probe mixture was applied to the slides and hybridized overnight in a moist chamber at 37 °C. The post-hybridization washes were performed as described in ‘LSI procedure’ (Abbott Molecular Inc., Des Plaines, IL, USA). Slides were counterstained with 125 ng/ml DAPI in antifade. A minimum of 25 tumor cells were analyzed for copy number changes of chromosomes 3, 7, 17 and 9p21 [22 ]. If no abnormalities were detected, the remaining cells were counted until a sufficient number of cells with chromosomal abnormalities were found, or until 200 cells were evaluated, or the whole specimen was analysed. A positive result was the presence of ≥4 cells with gains of two or more of chromosomes 3, 7 and 17. In the case of chromosome 9p21, a positive result was considered when ≥12 cells showed absence of 9p21 signals.
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