Specimen pretreatment method for reducing auto-fluorescence interference in FISH (fluorescence in situ hybridization) and reagent kit therefor

Abstract

The invention provides a specimen pretreatment method for reducing auto-fluorescence interference in FISH (fluorescence in situ hybridization) and a reagent kit therefor. The specimen pretreatment method comprises a first step of formalin fixation of the paraffin-embedded tissue, a second step of sectioning and drying, a third step of functioning of sodium thiocyanate, a fourth step of addition of 20[mu]l of collagenase type IV with the concentration of 5-10 mg / mL or elastinase with the concentration of 1mg / mL in the tissue with the area of 24*32mm and post-sealing functioning at the temperature of 37 DEG C for 0.5-1hr for degradation of the extracellular matrix with the auto-fluorescence characteristics, and a fifth step of functioning of pepsin at the temperature of 37 DEG C. The invention also provides a reagent kit for the specimen pretreatment method for reducing auto-fluorescence interference in FISH (fluorescence in situ hybridization). Through the specimen pretreatment based on the invention, the auto-fluorescence phenomena of the tissue is remarkably reduced, the ratio of fluorescent probe signals to background fluorescence is increased, and the detection quality and accuracy of FISH are improved.

Description

technical field [0001] The present invention provides a sample pretreatment method for reducing autofluorescence interference in fluorescence in situ hybridization, in particular, technically provides a method for fluorescence in situ hybridization (FISH, Fluorescence In Situ Hybridization) Add collagenase type IV or elastase to the sample pretreatment step to reduce the autofluorescence of human tissues and improve the resolution of fluorescent probe signals in fluorescence in situ hybridization . Background technique [0002] Fluorescence in situ hybridization (FISH) is a technique commonly used in clinical diagnosis, which can be used for early diagnosis, selection of treatment methods or monitoring of disease activity. For example, the treatment of breast cancer patients with trastuzumab is based on the detection of human epidermal growth factor receptor-2 (HER2) gene expression by fluorescence in situ hybridization (FISH) or when human epidermal growth factor is detect...

AI Technical Summary

Problems solved by technology

At present, commonly used fluorescent substances for labeling DNA or antibodies are FITC fluorescein isothiocyanate, Oregon green (Oregon green) 488, and Spectrum Green (SpectrumGreen). (endogenous fluorophores) such as: collagen, elastin, reduced nicotinamide adenine dinucleotide NADH, flavin mononucleotide (FMN, Flavin mononucleotide), flavin adenine dinucleotide (FAD, Flavin adenine dinucleotide) And aromatic amino acids (aromatic amino acids), if the specimen has strong background autofluorescence, it will directly affect the accuracy of fluorescence in situ hybridization and immunofluorescence staining interpretation. Such endogenous substances can also absorb similar Excitation light generated by short-wavelength light, resulting in background light generated by this type of autofluorescence will reduce the ratio of fluorescent probe or antibody signal to background fluorescence, resulting in poor resolution and quality of fluorescent signals, which will Affected signal counting and interpretation

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