CN103382499A - Specimen pretreatment method for reducing autofluorescence interference in fluorescence in situ hybridization method and reagent kit thereof - Google Patents

Abstract

A specimen pretreatment method for reducing autofluorescence interference in fluorescence in situ hybridization, including: Step 1: Formalin-fixed paraffin-embedded tissue; Step 2: Slicing and drying; Step 3: Thiocyanic acid Sodium effect; Step 4: Add 20 μl of type IV collagenase or 1 mg/mL elastase at a concentration of 5-10 mg/mL to a tissue with an area of 24×32 mm. After sealing, incubate at 37°C for 0.5-1 hr to degrade the autofluorescent properties. Extracellular matrix; Step 5: Use pepsin at 37°C. The present invention also provides a reagent set for specimen pretreatment that can be used to reduce autofluorescence interference in fluorescence in situ hybridization. Using the present invention for specimen pretreatment can significantly reduce tissue autofluorescence and improve fluorescent probes. The ratio of signal to background fluorescence improves the inspection quality and accuracy of fluorescence in situ hybridization.

Description

Specimen pretreatment method and reagent kit for reducing autofluorescence interference in fluorescence in situ hybridization

technical field

The present invention provides a sample pretreatment method for reducing autofluorescence interference in fluorescence in situ hybridization (FISH, Fluorescence In Situ Hybridization). The treatment step adds collagenase type IV or elastase treatment step to reduce the autofluorescence phenomenon of human tissue and improve the resolution of fluorescent probe signal by fluorescence in situ hybridization. the

Background technique

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 immunohistochemical staining (IHC) shows human epidermal growth factor receptor 2 (HER2) gene expression. Fluorescence in situ hybridization (FISH) was used for further confirmation when the HER22+ gene was detected, and patients who were more curative to the drug were screened out; -ALK) fusion gene-positive non-small cell lung adenocarcinoma (NSCLC) patients received anaplastic lymphoma kinase and hepatocyte growth factor receptor (ALK/C-met) dual-target small molecule inhibitors, Crizotinib ) treated patients can achieve more than 90% tumor shrinkage, and 57% of patients obtain objective remission. Therefore, it is effective to detect the fusion gene of echinoderm microtubule binding protein 4 and anaplastic lymphoma kinase (EML4-ALK) for clinical targets Therapy is of great help and benefit. the

Fluorescence in situ hybridization method is to separate the double-stranded deoxyribonucleic acid strands through denaturation in situ under the principle of not destroying the chain structure of deoxyribonucleic acid. The nucleic acid chains are hybridized and combined, and finally the fluorescent signal is directly observed under a fluorescent microscope to analyze whether there is a sequence corresponding to the deoxyribonucleic acid probe in the cell chromosome. At present, commonly used fluorescent substances for labeling deoxyribonucleic acid or antibodies are FITC fluorescein isothiocyanate, Oregon green (Oregon green) 488, and Spectrum Green (SpectrumGreen). ) 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 short-wavelength light to generate excitation The background light generated by such 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 affect signal counting and interpretation. the

Contents of the invention

The pretreatment step of the previous fluorescent in situ hybridization method used pepsin to break the peptide bond of the protein, but the lack of pepsin improved the phenomenon of autofluorescence of the sample. the

In order to improve the above problems, the present invention provides a sample pretreatment method for reducing autofluorescence interference in fluorescence in situ hybridization, comprising the following steps:

Step 1: formalin-fixed paraffin-embedded (FFPE) tissue;

Step 2: slice and bake;

Step 3: Sodium thiocyanate (NaSCN) effect;

Step 4: Add 20μl of 5-10mg/mL type IV collagenase or 1mg/mL elastase to the tissue with an area of 24×32mm, cover the slide and act for 0.5-1hr at 37°C to degrade extracellular cells with autofluorescent properties matrix; and

Step 5: Use pepsin to act at 37°C. the

In addition, the present invention provides two reagent kits for specimen pretreatment methods for reducing autofluorescence interference in fluorescence in situ hybridization. After 100mg of collagenase type IV was dissolved in 10mL of phosphate buffered saline (PBS, phosphate buffered saline), it was stored at a temperature of -20°C at a concentration of 10mg/mL; the other was taken from porcine pancreas (PORCINE PANCREAS) (≥4.0 units/mg protein) in the first type of pancreatic elastase (ELASTASE PANCREATIC TYPE I) 4mg dissolved in 1mL phosphate buffer solution (PBS, phosphate buffered saline), at a concentration of 4mg/mL at -20 ℃ store. the

The present invention adds type IV collagenase or elastase treatment step in the sample pretreatment step of fluorescence in situ hybridization (FISH) to degrade the extracellular matrix with endogenous fluorescent groups, which can significantly reduce the autofluorescence of human tissues phenomenon, improve the signal resolution of fluorescent probes, and improve the quality and accuracy of fluorescence in situ hybridization. the

The present invention will be described in further detail below in conjunction with specific embodiments. the

Description of drawings

Figure 1: Flow chart of the steps of the present invention. the

Fig. 2(A): A photo of a specimen blank that has not been treated by the method of the present invention is shown by a fluorescence microscope, and it can be seen that it has strong autofluorescence. the

Fig. 2(B): A photo of a blank specimen treated by the method of the present invention is shown by a fluorescence microscope, and its autofluorescence decreases. the

Fig. 3(A): Fluorescence in situ hybridization (FISH) of HER2/CEP17 after autofluorescent samples were pre-treated with pepsin by fluorescence microscope, the signal of FISH probe can be seen covered by background fluorescence. the

Fig. 3(B): A photo of fluorescence in situ hybridization of HER2/CEP17 after the sample with autofluorescence was treated by the method of the present invention through a fluorescence microscope, and the autofluorescence of the specimen can be seen to decrease. the

Figure 4(A): A fluorescent in situ hybridization (FISH) of HER2/CEP17 after the autofluorescent sample was pre-treated with pepsin was shown by a fluorescence microscope, and the signal of the probe was unclear and could not be counted. the

Fig. 4(B): A fluorescence in situ hybridization method of HER2/CEP17 after the autofluorescent sample was treated by the method of the present invention was shown by a fluorescence microscope after subtracting the background. It can be seen that the signal of the probe is clearly countable. the

Fig. 5(A): A photo of a blank piece of another specimen that has not been treated by the method of the present invention is shown by a fluorescence microscope, and it can be seen that it has strong autofluorescence. the

Fig. 5(B): A photo of a blank piece of another specimen treated by the method of the present invention is shown by a fluorescence microscope, and its autofluorescence can be seen to decrease. the

Figure 6(A): A fluorescent in situ hybridization (FISH) photo of EML4/ALK after pretreatment with pepsin was shown by a fluorescence microscope. It can be seen that the signal of the probe is unclear and cannot be counted. the

Fig. 6(B): A fluorescent in situ hybridization photograph of EML4/ALK after the autofluorescence sample was treated by the method of the present invention was shown by a fluorescence microscope, and the signal of the probe can be clearly counted. the

Detailed ways

Embodiments are carried out on the premise of the technical solutions of the present invention, and detailed implementation methods and specific operation processes are provided, but the protection scope of the present invention is not limited to the following embodiments. the

The methods used in the following examples are conventional methods unless otherwise specified. the

Referring to Figure 1, the present invention provides a sample pretreatment method for reducing autofluorescence interference in fluorescence in situ hybridization, comprising the following steps:

Step 1 (S1): formalin-fixed paraffin-embedded (FFPE) tissue;

Step 2 (S2): slicing and baking;

Step three (S3): sodium thiocyanate (NaSCN) effect;

Step 4 (S4): Add 20 μl of 5-10 mg/mL type IV collagenase or 1 mg/mL elastase to the tissue section with an area of 24×32 mm that has been treated as above, and seal the slice at 37°C for 0.5-1 hr to degrade extracellular matrix with autofluorescent properties; and

Step 5 (S5): Use pepsin to act at 37°C. the

In the pretreatment method of the above-mentioned specimen, in the first step (S1), the tissue is various tissues such as breast cancer tissue, lung cancer tissue, skin tissue, kidney tissue, etc. The method is: soak and fix the tissue in 10% neutral buffered formalin (10% neutral buffered formalin) for a minimum of 6 hours and a maximum of 48 hours, and then dehydrate with alcohol (use alcohol first) After reacting for 3 hours with a concentration of 95wt%, and then using an alcohol concentration of 99.8wt% for 3 hours), soak in xylene (Xylene) for 2 hours to make it transparent, and finally use paraffin (Paraffin) to embed (about 3 hours). the

In the second step (S2), the specific method of slicing is: place the wax block on the knife seat of the tissue slicer to clamp the wax block, cut 4 μm into cold water with the tissue slicer, and remove the slice with a glass slide and move to 50°C. Flatten the tissue slices in a water bath at ℃. After the tissue slices are flattened, pick up the slices again with a glass slide to attach the slices to the slides. Then stand the slides upright so that the residual water in the slices flows down. Shake it out vigorously; the specific method of drying the slices is to place the slices in an oven at 56°C and bake for at least 2 hours or overnight. the

In the third step (S3), the baked slices are put into a sodium thiocyanate (NaSCN) solution with a concentration of 1M, and heated at 80°C for 15-25min. The purpose of treatment with sodium thiocyanate (NaSCN) is to assist in unwinding deoxyribonucleic acid (DNA) or protein intertwining caused by formalin fixation. the

In the fifth step (S5), the mass concentration of pepsin (pepsin) is 0.005%. After the fourth step, it is added to the tissue section and placed at 37° C. for 3-10 minutes. The purpose of treating with pepsin is to break the peptide bond of the protein, degrade the protein, reduce the phenomenon of autofluorescence produced by the intact protein, and make it easier for the fluorescent probe to bind to the chromosome. the

The present invention pre-treats various wax block tissues with an appropriate proportion of collagenase type IV and elastase, which can degrade the extracellular matrix with endogenous fluorescent groups, and can significantly reduce the autologous Fluorescence phenomenon, reducing the background signal of fluorescence detection, improving the signal resolution of fluorescent probes, and improving the quality and accuracy of fluorescence in situ hybridization. Please refer to Fig. 2(A). Fig. 2(A) was observed by fluorescence microscope with fluorescein isothiocyanate filter (spectra green FITC filter), without collagenase type IV or elastase (Elastase) The edge of the sample treated by elastase presents a bright white edge as a part of fluorescence, showing that the blank sheet of the sample has strong autofluorescence; after being processed by the sample pretreatment method of the present invention, the sample can be seen from Figure 2 (B) The part of the edge fluorescence has turned gray, indicating that the autofluorescence of the blank specimen has decreased. Figure 3(A) shows that autofluorescent samples were not treated with collagenase type IV or elastase, but were only pre-treated with pepsin, and the human epidermal growth factor receptor-2 And the fluorescent in situ hybridization method of chromosome 17 centromere (HER2/CEP17), under the fluorescence microscope, the original signal map has a strong background, and the gray cluster in the picture indicates that the background fluorescence has covered the fluorescent in situ hybridization probe signal; However, Fig. 3(B) shows that after the sample pretreatment method of the present invention, the autofluorescence of the sample decreases, and the gray clusters in the figure are significantly reduced, indicating that the background fluorescence is significantly reduced, and the white points in the figure are clearly visible, which can provide calculation The number of signal points represents a significant increase in the signal resolution and countable number of signal points of the fluorescent in situ hybridization method. See also Figure 4(A) for human epidermal growth factor after pepsin pretreatment only without collagenase type IV or elastase treatment in samples with autofluorescence Receptor-2 and chromosome 17 centromere (HER2/CEP17) fluorescence in situ hybridization method, under the fluorescence microscope observation, the signal of the probe is unclear after deducting the background, and cannot be counted; and Fig. 4 (B) shows that after the method of the present invention After the sample pretreatment method, the autofluorescence of the sample decreased, the background fluorescence decreased significantly, and the signal resolution and number of countable signal points of the fluorescence in situ hybridization method increased significantly. The white points in the figure are clearly visible, which can provide calculation The number of signal points means that the probe signal is clear and countable after subtracting the background. Please refer to Fig. 5(A), by observing with a FITC filter under a fluorescence microscope, the detected samples without collagenase type IV or elastase treatment The bright white edge present on the edge of the object is the fluorescent part, and the sample blank has strong autofluorescence; after being processed by the sample pretreatment method of the present invention, it can be seen from Fig. 5 (B) that the fluorescent part of the edge of the sample has appeared Gray indicates that the autofluorescence of the blank piece of the specimen is decreased. Fig. 6(A) also shows that samples with autofluorescence were not treated with collagenase type IV or elastase, but only pre-treated with pepsin, and the echinoderm microtubule-binding protein 4 Fluorescent in situ hybridization with anaplastic lymphoma kinase (EML4-ALK), observed under a fluorescence microscope, the original signal pattern has a strong background, and the gray cluster in the image indicates that the background fluorescence has covered the signal of the fluorescent in situ hybridization probe, after subtracting the background The probe signal is unclear and cannot be counted; Figure 6(B) shows that after the sample pretreatment method of the present invention, the autofluorescence of the sample decreases, and the gray clusters in the figure are significantly reduced, indicating that the background fluorescence is significantly reduced. The white dots are clearly visible and can be used to calculate the number of signal points, which means that the background fluorescence is significantly reduced. The signal resolution and number of countable signal points of the fluorescent in situ hybridization method are significantly increased. After the background is subtracted, the probe signal is clear and countable. the

The present invention also provides two reagent sets that can be used in the above-mentioned sample pretreatment method for reducing autofluorescence interference in fluorescence in situ hybridization, one of which is produced by 100% pure Clostridium histolyticum (clostridium histolyticum) After 100mg of collagenase type IV was dissolved in 10mL of phosphate buffered saline (PBS, phosphate buffered saline), it was stored at a concentration of 10mg/mL at -20°C; another reagent was obtained from porcine pancreas (PORCINE PANCREAS) (≥4.0 units/mg protein) in the first type of pancreatic elastase (ELASTASE PANCREATIC TYPE I) 4mg dissolved in 1mL phosphate buffer (PBS, phosphate buffered saline), with a concentration of 4mg/mL at -20 Store at a temperature of °C. The use of this reagent for pretreatment of specimens can also improve the limitations and shortcomings of the current fluorescence in situ hybridization test kits, and achieve the same effect as the above pretreatment method. Therefore, the present invention can be applied to any autofluorescence phenomenon. Specimen types (such as breast cancer, lung cancer, skin, kidney, etc.). the

The reagents of the present invention can be formulated with appropriate concentration and action time according to different specimens. For example, when used in the pretreatment of mammary gland tissue specimens, the above-mentioned type IV collagenase reagent can be used at an action concentration of 5-10 mg/mL. The temperature is 37°C, and it acts at 30-60 minutes; or when it is used in the pretreatment of lung tissue samples, the above-mentioned elastase reagent can be used at an action concentration of 1 mg/mL, at an action temperature of 37°C, and at 30-60 minutes effect. the

In summary, the present invention can improve the problem that the specimen pretreatment step with pepsin in the existing fluorescence in situ hybridization method cannot reduce the autofluorescence phenomenon of the specimen. According to the results of parallel experiments, the specimen with strong autofluorescence , after being treated by the method of the present invention, the autofluorescence of the sample is significantly and greatly reduced. Compared with the pepsin treatment method, the signal of the probe of the sample can be clearly counted after the treatment of the present invention, and the resolution of the probe signal is greatly and significantly improved. the

Claims (3)

1. A sample pretreatment method for reducing autofluorescence interference in fluorescence in situ hybridization, comprising the following steps: Step 1: formalin-fixed paraffin-embedded (FFPE) tissue; Step 2: slice and bake; Step 3: Sodium thiocyanate (NaSCN) effect; Step 4: Add 20μl of 5-10mg/mL type IV collagenase or 1mg/mL elastase to the tissue with an area of 24×32mm, cover the slide and act for 0.5-1hr at 37°C to degrade extracellular cells with autofluorescent properties matrix; and Step 5: Use pepsin to act at 37°C. 2. A reagent kit for the method as claimed in claim 1, wherein 100 mg of collagenase type IV (collagenase type IV) produced by clostridium histolyticum (clostridium histolyticum) with a purity of 100% is dissolved in 10 mL phosphate buffered saline (PBS, phosphate buffered saline), stored at a concentration of 10 mg/mL at -20°C. 3. A reagent kit for the method as claimed in claim 1, which is obtained from the first type pancreatic elastase (ELASTASE PANCREATIC TYPE I) 4mg in porcine pancreas (PORCINE PANCREAS) (≥4.0 units/mg protein) After dissolving in 1 mL of phosphate buffered saline (PBS, phosphate buffered saline), store at a concentration of 4 mg/mL at -20°C. the

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