CN110865056A - Preservation method of transgenic zebra fish protein fluorescence - Google Patents
Preservation method of transgenic zebra fish protein fluorescence Download PDFInfo
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- CN110865056A CN110865056A CN201911076045.6A CN201911076045A CN110865056A CN 110865056 A CN110865056 A CN 110865056A CN 201911076045 A CN201911076045 A CN 201911076045A CN 110865056 A CN110865056 A CN 110865056A
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- methanol
- fluorescence
- transgenic zebrafish
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- deionized water
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
Abstract
The invention discloses a method for preserving transgenic zebra fish protein fluorescence, which comprises the following steps: collecting transgenic zebrafish embryos, and placing the transgenic zebrafish embryos in fresh 4% PFA for 2-6 hours at room temperature or 12-24 hours at 4 ℃; washing with 0.1M glycine/0.05% -1% DMSO/0.1% -0.5% triton for three times, each for 5-15 minutes; dehydrating with 25%, 50%, 75% and 100% methanol in sequence, each time for 5-10 min; storage at-20 ℃ for up to several weeks; when in observation, the sample is washed by 75 percent, 50 percent and 25 percent of methanol in sequence, and each time lasts for 5 to 10 minutes; PBST is washed for three times, 5-15 minutes each time; and (5) observing and photographing under a fluorescence microscope. According to the invention, antibodies and immunofluorescence staining are not needed, so that the cost and the time are saved, subsequent experiments or observation can be carried out at any time when needed, repeated labor is reduced, the experiment period is obviously shortened, and the cost is saved.
Description
Technical Field
The invention belongs to the field of biochemistry, and particularly relates to a preservation method of transgenic zebra fish protein fluorescence, which is used for prolonging the preservation time of the transgenic zebra fish protein fluorescence.
Background
There have been two common methods for observing tissue proteins, but each has its disadvantages:
firstly, immunofluorescent staining is carried out by using an antibody, and the disadvantages that ① immunofluorescent staining requires the use of an expensive antibody, the requirements on the technology for selection, storage and use of the antibody are high, and ② from tissue fixation to staining completion requires at least 4-5 days and takes a long time.
Secondly, the specific protein is fluorescently labeled by using a transgenic technology, and the defects that ① transgenic zebra fish is more precious and the model is more difficult to construct, and ② timeliness is caused, the fluorescence observation of the transgenic zebra fish protein can be only carried out at a corresponding time point, and is irreversible once the fluorescence observation is missed.
Disclosure of Invention
The purpose of the invention is as follows: in order to solve the defects of the prior art, the invention provides a method for preserving fluorescence of transgenic zebra fish protein.
The technical scheme is as follows: a method for preserving fluorescence of transgenic zebra fish protein comprises the following steps:
(1) collecting transgenic zebrafish embryos, and placing the transgenic zebrafish embryos in fresh 4% PFA for 2-6 hours at room temperature or 12-24 hours at 4 ℃;
(2) washing with 0.1M glycine/0.05% -1% DMSO/0.1% -0.5% triton for three times, each for 5-15 minutes;
(3) dehydrating with 25%, 50%, 75% and 100% methanol in sequence, each time for 5-10 min;
(4) storage at-20 ℃ for up to several weeks;
(5) when in observation, the sample is washed by 75 percent, 50 percent and 25 percent of methanol in sequence, and each time lasts for 5 to 10 minutes;
(6) PBST is washed for three times, 5-15 minutes each time;
(7) and (5) observing and photographing under a fluorescence microscope.
As an optimization: the 4% PFA comprises the following components in parts by weight: 4g of Paraformaldehyde (PFA); na (Na)2HPO4·12H2O：0.475g；NaH2PO4·2H2O: 2.9 g; the above components were dissolved in 100ml of deionized water, adjusted to pH 7.3 with NaOH, filtered and kept at 4 ℃ until needed.
As an optimization: the 0.1M glycine/0.05% -1% DMSO/0.1% -0.5% triton comprises the following components in parts by weight: 0.7507g of glycine; DMSO, DMSO: 0.05-1 mL; triton: 0.02-0.1 mL; 0.1M PBS was added to 100 ml.
As an optimization: the 0.1M PBS comprisesThe following components in proportion: NaCl 0.8 g; NaH2PO4·2H2O：0.296g；Na2HPO4·12H2O: 2.9 g; the above components were dissolved in 100mL of deionized water, adjusted to pH 7.3 with NaOH, filtered and kept at 4 ℃ until needed.
As an optimization: the 25% methanol is as follows: 12.5mL of methanol, 37.5mL of deionized water.
As an optimization: the 50% methanol is: 25mL of methanol and 25mL of deionized water.
As an optimization: the 75% methanol is: 37.5mL of methanol, 12.5mL of deionized water.
As an optimization: the PBST comprises the following components in proportion: 0.1M PBS: 10 mL; tween-20: 0.1 mL; deionized water: 89.9 mL.
Has the advantages that: the invention does not need to use antibody and carry out immunofluorescence staining, thereby saving the cost and time. And if the protein fluorescence of the transgenic fish can be stored for a long time, subsequent experiments or observation can be carried out at any time when needed, the repeated labor is reduced, the experiment period is obviously shortened, and the cost is saved.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below so that those skilled in the art can better understand the advantages and features of the present invention, and thus the scope of the present invention will be more clearly defined. The embodiments described herein are only a few embodiments of the present invention, rather than all embodiments, and all other embodiments that can be derived by one of ordinary skill in the art without inventive faculty based on the embodiments described herein are intended to fall within the scope of the present invention.
Examples
A method for preserving fluorescence of transgenic zebra fish protein comprises the following steps:
(1) collecting transgenic zebrafish embryos, and placing the transgenic zebrafish embryos in fresh 4% PFA for 2-6 hours at room temperature or 12-24 hours at 4 ℃; wherein, the 4% PFA comprises the following components in proportion: 4g of Paraformaldehyde (PFA); na (Na)2HPO4·12H2O：0.475g；NaH2PO4·2H2O: 2.9 g; the above components were dissolved in 100ml of deionized water, adjusted to pH 7.3 with NaOH, filtered and kept at 4 ℃ until needed.
(2) Washing with 0.1M glycine/0.05% -1% DMSO/0.1% -0.5% triton for three times, each for 5-15 minutes; wherein, the 0.1M glycine/1% DMSO/0.1% triton comprises the following components in proportion: 0.7507g of glycine; DMSO, DMSO: 0.05-1 mL; triton: 0.02-0.1 mL; 0.1M PBS was added to 100 ml.
The 0.1M PBS comprises the following components in proportion: NaCl 0.8 g; NaH2PO4·2H2O：0.296g；Na2HPO4·12H2O: 2.9 g; the above components were dissolved in 100mL of deionized water, adjusted to pH 7.3 with NaOH, filtered and kept at 4 ℃ until needed.
(3) Dehydrating with 25%, 50%, 75%, and 100% methanol for 5-10 min each time.
(4) Storage at-20 ℃ for several weeks.
(5) When observing, washing with 75%, 50% and 25% methanol for 5-10 minutes each time.
(6) PBST is washed for three times, 5-15 minutes each time; wherein, the PBST comprises the following component distribution ratio: 0.1 MPBS: 10 mL; tween-20: 0.1 mL; deionized water: 89.9 mL.
(7) And (5) observing and photographing under a fluorescence microscope.
In this embodiment, the 25% methanol is: 12.5mL of methanol, 37.5mL of deionized water. The 50% methanol is: 25mL of methanol and 25mL of deionized water. The 75% methanol is: 37.5mL of methanol, 12.5mL of deionized water.
The invention does not need to use antibody and carry out immunofluorescence staining, thereby saving the cost and time. And if the protein fluorescence of the transgenic fish can be stored for a long time, subsequent experiments or observation can be carried out at any time when needed, the repeated labor is reduced, the experiment period is obviously shortened, and the cost is saved.
Claims (8)
1. A method for preserving fluorescence of transgenic zebra fish protein is characterized in that: the method comprises the following steps:
(1) collecting transgenic zebrafish embryos, and placing the transgenic zebrafish embryos in fresh 4% PFA for 2-6 hours at room temperature or 12-24 hours at 4 ℃;
(2) washing with 0.1M glycine/0.05% -1% DMSO/0.1% -0.5% triton for three times, each for 5-15 minutes;
(3) dehydrating with 25%, 50%, 75% and 100% methanol in sequence, each time for 5-10 min;
(4) storage at-20 ℃ for up to several weeks;
(5) when in observation, the sample is washed by 75 percent, 50 percent and 25 percent of methanol in sequence, and each time lasts for 5 to 10 minutes;
(6) PBST is washed for three times, 5-15 minutes each time;
(7) and (5) observing and photographing under a fluorescence microscope.
2. The method for preserving fluorescence of transgenic zebrafish according to claim 1, characterized in that: the 4% PFA comprises the following components in percentage by weight: 4g of Paraformaldehyde (PFA); na (Na)2HPO4·12H2O：0.475g；NaH2PO4·2H2O: 2.9 g; the above components were dissolved in 100ml of deionized water, adjusted to pH 7.3 with NaOH, filtered and kept at 4 ℃ until needed.
3. The method for preserving fluorescence of transgenic zebrafish according to claim 1, characterized in that: the 0.1M glycine/0.05% -1% DMSO/0.1% -0.5% triton comprises the following components in parts by weight: 0.7507g of glycine; DMSO, DMSO: 0.05-1 mL; triton: 0.02-0.1 mL; 0.1M PBS was added to 100 ml.
4. The method for preserving fluorescence of transgenic zebrafish according to claim 1, characterized in that: the 0.1MPBS comprises the following components in proportion: NaCl 0.8 g; NaH2PO4·2H2O：0.296g；Na2HPO4·12H2O: 2.9 g; the above components were dissolved in 100mL of deionized water, adjusted to pH 7.3 with NaOH, filtered and kept at 4 ℃ until needed.
5. The method for preserving fluorescence of transgenic zebrafish according to claim 1, characterized in that: the 25% methanol is as follows: 12.5mL of methanol, 37.5mL of deionized water.
6. The method for preserving fluorescence of transgenic zebrafish according to claim 1, characterized in that: the 50% methanol is: 25mL of methanol and 25mL of deionized water.
7. The method for preserving fluorescence of transgenic zebrafish according to claim 1, characterized in that: the 75% methanol is: 37.5mL of methanol, 12.5mL of deionized water.
8. The method for preserving fluorescence of transgenic zebrafish according to claim 1, characterized in that: the PBST comprises the following components in proportion: 0.1M PBS: 10 mL; tween-20: 0.1 mL; deionized water: 89.9 mL.
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Citations (7)
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| US6299858B1 (en) * | 1998-02-23 | 2001-10-09 | Phylonix Pharmaceuticals, Inc. | Methods of screening agents for activity using teleosts |
| US20050005317A1 (en) * | 2003-07-01 | 2005-01-06 | Hwang Sheng-Ping L. | Expression of zebrafish bone morphogenetic protein 4 |
| CN1866025A (en) * | 2006-06-01 | 2006-11-22 | 中国科学院海洋研究所 | Method for detecting activity of anti-angiogenesis protein factor or pro-angiogenesis protein factor via zebra fish embryo model and application method thereof |
| CN105918255A (en) * | 2016-06-02 | 2016-09-07 | 贵州医科大学 | Transgenic zebrafish model with G6PD (gluocose-6-phosphate dehydrogenase deficiency) 118-144 site defects |
| US20180282689A1 (en) * | 2017-03-28 | 2018-10-04 | The Board Of Trustees Of The Leland Stanford Junior University | Assembly of functionally integrated human forebrain spheroids and methods of use thereof |
| CN110055277A (en) * | 2018-11-09 | 2019-07-26 | 华南理工大学 | A kind of transgenic zebrafish model of liver specificity aggregation macrophage |
| WO2019164473A1 (en) * | 2018-02-20 | 2019-08-29 | Visikol Inc. | Method for visualization of 3-d tissue cultures |
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2019
- 2019-11-06 CN CN201911076045.6A patent/CN110865056A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6299858B1 (en) * | 1998-02-23 | 2001-10-09 | Phylonix Pharmaceuticals, Inc. | Methods of screening agents for activity using teleosts |
| US20050005317A1 (en) * | 2003-07-01 | 2005-01-06 | Hwang Sheng-Ping L. | Expression of zebrafish bone morphogenetic protein 4 |
| CN1866025A (en) * | 2006-06-01 | 2006-11-22 | 中国科学院海洋研究所 | Method for detecting activity of anti-angiogenesis protein factor or pro-angiogenesis protein factor via zebra fish embryo model and application method thereof |
| CN105918255A (en) * | 2016-06-02 | 2016-09-07 | 贵州医科大学 | Transgenic zebrafish model with G6PD (gluocose-6-phosphate dehydrogenase deficiency) 118-144 site defects |
| US20180282689A1 (en) * | 2017-03-28 | 2018-10-04 | The Board Of Trustees Of The Leland Stanford Junior University | Assembly of functionally integrated human forebrain spheroids and methods of use thereof |
| WO2019164473A1 (en) * | 2018-02-20 | 2019-08-29 | Visikol Inc. | Method for visualization of 3-d tissue cultures |
| CN110055277A (en) * | 2018-11-09 | 2019-07-26 | 华南理工大学 | A kind of transgenic zebrafish model of liver specificity aggregation macrophage |
Non-Patent Citations (2)
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| 刘鑫 等: "视黄酸信号通路调控咽弓神经经嵴影响斑马鱼牙齿发育的研究", 《华西口腔医学杂志》 * |
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Application publication date: 20200306 |