FISH pretreatment method and pretreatment fluid for FFPE sample

A pretreatment solution and pretreatment technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of serious fluorescence and background, poor FFPE effect of liver cancer, etc., and achieve the effect of reducing fluorescence and background

Active Publication Date: 2015-11-04
AMOY DIAGNOSTICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0018] However, this commonly used pretreatment method, firstly, the FFPE fluorescence and background after treatment are still relatively serious; secondly, it is...

Method used

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  • FISH pretreatment method and pretreatment fluid for FFPE sample
  • FISH pretreatment method and pretreatment fluid for FFPE sample
  • FISH pretreatment method and pretreatment fluid for FFPE sample

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] (1) Bake slices: place the slides on a 56°C oven and bake the slides for 15 minutes.

[0065] (2) Dewaxing: In a fume hood, immerse slides in 3 cylinders of xylene in sequence, 10 min each time.

[0066] (3) Removal of residual xylene: immerse the slides in 3 cylinders of absolute ethanol in turn, shake a few times each time to remove the xylene.

[0067] (4) Hydration: immerse slides in absolute ethanol, 85% ethanol, 70% ethanol, and deionized water in sequence, for 1 min each time.

[0068] (5) Treatment with pretreatment solution I (60 mM, pH 7.0 EDTA solution): immerse slides (tissue side up) in boiling pretreatment solution I, and boil for 20 minutes steadily.

[0069] (6) Immerse the slide in deionized water and wash for 1 min.

[0070] (7) Immerse the slide in PBS solution and wash for 1 min.

[0071] (8) Treatment with pretreatment solution II (0.05% pontamine sky blue): immerse slides in pretreatment solution II for 10 minutes. Shake and mix the Pretreatmen...

Embodiment 2

[0084] (1) Bake slices: place the slides on a 56°C baker for 30 minutes.

[0085] (2) Dewaxing: In a fume hood, immerse slides in 3 cylinders of xylene in sequence, 10 min each time.

[0086] (3) Removal of residual xylene: immerse the slides in 3 cylinders of absolute ethanol in turn, shake a few times each time to remove the xylene.

[0087] (4) Hydration: immerse slides in absolute ethanol, 85% ethanol, 70% ethanol, and deionized water in sequence, for 1 min each time.

[0088] (5) Treatment with pretreatment solution I (10 mM citrate buffer solution): immerse the glass slide (tissue side facing up) in the boiling pretreatment solution I, and cook for 20 minutes steadily.

[0089] (6) Immerse the slide in deionized water and wash for 1 min.

[0090] (7) Immerse the slide in PBS solution and wash for 1 min.

[0091] (8) Treatment with pretreatment solution II (0.05% trypan blue): immerse the glass slide in pretreatment solution II and treat for 15 minutes. Shake and mix ...

Embodiment 3

[0104] (1) Bake slices: place the slides on a 56°C baker for 25 minutes.

[0105] (2) Dewaxing: In a fume hood, immerse slides in 3 cylinders of xylene in sequence, 10 min each time.

[0106] (3) Removal of residual xylene: immerse the slides in 3 cylinders of absolute ethanol in turn, shake a few times each time to remove the xylene.

[0107] (4) Hydration: immerse slides in absolute ethanol, 85% ethanol, 70% ethanol, and deionized water in sequence, for 1 min each time.

[0108] (5) Treatment with pretreatment solution I (5% sodium thiocyanate solution): immerse the glass slide (with the tissue side facing up) in the boiling pretreatment solution I, and boil for 15 minutes steadily.

[0109] (6) Immerse the slide in deionized water and wash for 1 min.

[0110] (7) Immerse the slide in PBS solution and wash for 1 min.

[0111] (8) Treatment with pretreatment solution II (0.05% trypan blue): immerse the glass slide in pretreatment solution II and treat for 15 minutes. Shak...

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Abstract

The invention discloses a FISH pretreatment method and pretreatment fluid for an FFPE sample. The pretreatment fluid comprises a pretreatment fluid I, a pretreatment fluid II and a pretreatment fluid III, wherein, the pretreatment fluid I is at least one selected from the group consisting of an EDTA solution, a citric acid buffer and a sodium sulfocyanate solution, the pretreatment fluid II is at least one selected from the group consisting of pontamine sky blue with a concentration of 0.01 to 0.5% and trypan blue with a concentration of 0.025 to 1%, the pretreatment fluid III is sodium borohydride with a concentration of 0.1 to 5%, and the above-mentioned substances are weighed according to a mass volume ratio. The pretreatment method is composed of a plurality of steps. The FISH pretreatment method and pretreatment fluid provided by the invention can substantially reduce FISH autofluorescence and background of liver cancer FFPE.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a FISH pretreatment method for liver cancer tissue paraffin section (FFPE) samples. Background technique [0002] Autofluorescence is a common interference factor in related studies such as fluorescein labeling, usually caused by endogenous riboflavin (Flavin), reduction of NAD(P)H, lipofuscin (Lipofuscin), reticular fibers, collagen and elastin protein caused. In tissue cells with vigorous metabolism such as the liver and kidney, there are a large number of NADP-NADPH conversion phenomena, and a lot of riboflavin and lipofuscin have accumulated at the same time; in its abundant blood vessels, the content of collagen and elastin is also high, while in the liver Mostly reticular fibers. Such molecules have strong autofluorescence and background under a fluorescence microscope, which seriously affects the results of fluorescence in situ hybridization (FISH) of liver tissue. Therefor...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6806C12Q2521/537
Inventor 卢皇彬林清华唐郑华纪斌峰宋庆涛阮力
Owner AMOY DIAGNOSTICS CO LTD
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