Hybrid buffer solution system and in situ hybridization method suitable for bacteria FISH (Fluorescence in situ hybridization) detection

A hybridization buffer and fluorescence in situ hybridization technology, applied in the field of molecular hybridization detection, can solve the problems of many components and poor results, and achieve the effect of low background, low cost and high specificity of results

Pending Publication Date: 2020-01-31
武汉赛维尔生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The hybridization solutions used in bacterial FISH in current literature generally contain ingredients such as dextran sulfate, dithiothreitol, and salmon sperm DNA. The product has many ingredients and the effect is not good.

Method used

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  • Hybrid buffer solution system and in situ hybridization method suitable for bacteria FISH (Fluorescence in situ hybridization) detection
  • Hybrid buffer solution system and in situ hybridization method suitable for bacteria FISH (Fluorescence in situ hybridization) detection
  • Hybrid buffer solution system and in situ hybridization method suitable for bacteria FISH (Fluorescence in situ hybridization) detection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] The proportion of each component in the hybridization buffer is: 15% deionized formamide, 2×SSC, 0.5M EDTA, 5×Denhardt, 50mMNaH 2 PO 4 .

[0027] The method for carrying out bacterial FISH in situ hybridization using the hybridization buffer comprises the steps:

[0028] (1) Tissue slices were fixed, dewaxed and washed with water: the intestinal tissue slices were fixed with 4% paraformaldehyde, and then the tissue slices were placed in xylene I for 15 min, xylene II for 15 min, absolute ethanol I for 5 min, absolute ethanol II for 5 min, 85% (volume concentration) alcohol for 5 minutes, 75% (volume concentration) alcohol for 5 minutes, washed with DEPC;

[0029] (2) Permeabilization and digestion: according to the length of tissue fixation, place the slices in 1× repair solution (repair solution: DEPC and water diluted at 1:19) at 90°C for 5-10 minutes, and cool naturally; post-gene strokes According to the characteristics of different indicators of different tissue...

Embodiment 2

[0039] The proportion of each component in the hybridization buffer is: 15% deionized formamide, 2×SSC, 0.5M EDTA, 5×Denhardt, 50mMNaH 2 PO 4 .

[0040] The method for carrying out bacterial FISH in situ hybridization using the hybridization buffer comprises the steps:

[0041] (1) Tissue slices were fixed, dewaxed and washed with water: the lung tissue slices were fixed with 4% paraformaldehyde, and then the tissue slices were placed in xylene I for 15 min, xylene II for 15 min, absolute ethanol I for 5 min, absolute ethanol II for 5 min, 85 % (volume concentration) alcohol 5min, 75% (volume concentration) alcohol 5min, DEPC washing;

[0042](2) Permeabilization and digestion: according to the length of tissue fixation, place the slices in 1× repair solution (repair solution: DEPC and water diluted at 1:19) at 90°C for 5-10 minutes, and cool naturally; post-gene strokes According to the characteristics of different indicators of different tissues, drop proteinase K working...

Embodiment 3

[0051] The proportion of each component in the hybridization buffer is: 15% deionized formamide, 2×SSC, 0.5M EDTA, 5×Denhardt, 50mMNaH 2 PO 4 .

[0052] The method for carrying out bacterial FISH in situ hybridization using the hybridization buffer comprises the steps:

[0053] (1) Tissue slices were fixed, dewaxed and washed with water: the lung tissue slices were fixed with 4% paraformaldehyde, and then the tissue slices were placed in xylene I for 15 min, xylene II for 15 min, absolute ethanol I for 5 min, absolute ethanol II for 5 min, 85 % (volume concentration) alcohol 5min, 75% (volume concentration) alcohol 5min, DEPC washing;

[0054] (2) Permeabilization and digestion: according to the length of tissue fixation, place the slices in 1× repair solution (repair solution: DEPC and water diluted at 1:19) at 90°C for 5-10 minutes, and cool naturally; post-gene strokes According to the characteristics of different indicators of different tissues, drop proteinase K workin...

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Abstract

The invention belongs to the technical field of molecular hybridization detection, and particularly relates to a hybrid buffer solution system and in situ hybridization method suitable for bacteria FISH (Fluorescence in situ hybridization) detection. The hybrid buffer solution comprises the following ingredients at a ratio: 15% deionized formamide, 2*SSC, 0.5M EDTA (Ethylene Diamine Tetraacetic Acid), 5*Denhardt and 50mM NaH2PO4. The hybrid buffer solution disclosed by the invention is especially suitable for bacteria FISH detection. The hybrid buffer solution has the advantages of high resultspecificity, low result background, convenience in use, low cost and the like when the hybrid buffer solution is used for carrying out FISH hybridization.

Description

technical field [0001] The invention belongs to the technical field of molecular hybridization detection, and in particular relates to a hybridization buffer system suitable for bacterial FISH detection and an in-situ hybridization method thereof. Background technique [0002] The FISH experiment uses the mechanical principle of complementary base pairing to pair the specific labeled probe sequence with the target gene fragment, and then displays the position and quantity of the binding point through fluorescence or chemical reaction. [0003] Nucleotide probes are a general tool for detecting RNA in tissue cells in in situ hybridization, usually including cRNA, cDNA and oligonucleotide probes. The synthesis of oligonucleotide probes is simpler and more convenient than the other two, and the cost is low. Specific oligonucleotide probes need to control the probe to only bind to the target sequence under a specific hybridization environment. Here, the tool of hybridization s...

Claims

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Application Information

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Patent Type & AuthorityApplications(China)
IPC IPC(8): C12Q1/6841
CPCC12Q1/6841C12Q2527/125C12Q2563/107
Inventor李绪易婷婷陈立娟张高英
Owner武汉赛维尔生物科技有限公司