CN111474358A - 3D (three-dimensional) immunofluorescence staining kit and application thereof - Google Patents
3D (three-dimensional) immunofluorescence staining kit and application thereof Download PDFInfo
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Abstract
The invention provides a 3D (three-dimensional) immunofluorescence staining kit and application thereof, wherein the kit comprises a fixing solution, a cell membrane punching solution, a sealing solution, a PBS (phosphate buffer solution), a primary antibody, a fluorescent secondary antibody, a dehydrating agent and ion exchange resin, wherein the cell membrane punching solution is prepared by adding 2.5% of cane sugar, 20% of DMSO, 5% of Tween-20 and 5% of Triton X-100 into 0.1M of PBS solution and fully stirring and dissolving the mixture. The kit and the immunofluorescence staining method can be used for overall fluorescence labeling of large tissues, and compared with the traditional immunofluorescence staining, the method can be used for improving the protein positioning of a certain layer in the tissues to the 3D protein positioning labeling, so that more accurate absolute quantitative analysis is realized.
Description
Technical Field
The invention relates to the field of immunofluorescence staining, and particularly relates to a 3D (three-dimensional) immunofluorescence staining kit and application thereof.
Background
The immunofluorescence staining technique is a protein molecular detection technique developed by combining a biochemical technique and a microscopic technique based on immunology. The immunofluorescence technology utilizes the principle that an antigen is highly specifically combined with an antibody, couples a certain measurable substance to the antibody or the antigen, and qualitatively, quantitatively detects the antigen or the antibody to be detected in a sample by detecting the existence and the content of a marker. Coons, et al, 1941 succeeded for the first time in antigen localization using fluorescein labeled antibodies. Particularly, in recent thirty years, the immunofluorescence technology is the fastest-developing and most widely-applied technology in the labeling immunity technology, and has the advantages of strong specificity, high sensitivity, high detection speed, convenience in observation and the like. Immunofluorescence staining and imaging analysis are indispensable detection techniques for studying cell tissue morphology and in situ antigen expression, and are widely applied to various fields of clinical pathological diagnosis and medical and biological research.
The traditional immunofluorescence staining method is a method of using a common paraffin section to mark antigen reaction in tissues by using antibodies and then using fluorescein for color development. The sample dyed by immunofluorescence usually contains multidimensional biological information, including tissue structure information, spatial distribution information of different cell types, co-expression information of different signal path proteins or cell function markers and the like, and the complete recording and full interpretation of the information are of great significance for researching the mechanism of disease occurrence and development and discussing the effective method for diagnosing and treating diseases. However, the traditional immunofluorescence staining method has the great defects that only a certain layer of protein location can be marked, and the judgment of the immunofluorescence image mostly adopts a semi-quantitative analysis mode of observing the proportion and distribution of positive staining areas by manual counting, so that the analysis is greatly limited; even if continuous slice imaging is used, due to the limitation and loss of slice thickness, even two continuous slices are difficult to achieve nanoscale accurate alignment, the continuity of cells cannot be maintained, and single-cell-level co-localization analysis cannot be performed.
Disclosure of Invention
In view of the above, the invention provides a 3D immunofluorescence staining kit and applications thereof, which can realize whole fluorescence labeling of large tissues and perform data analysis of three-dimensional imaging.
The technical scheme of the invention is realized as follows:
in one aspect, the invention provides a 3D (three-dimensional) immunofluorescence staining kit, which comprises a fixing solution, a cell membrane punching solution, a sealing solution, a PBS (phosphate buffer solution), a primary antibody, a fluorescent secondary antibody, a dehydrating agent and ion exchange resin, wherein the fixing solution is at least one of ethanol, acetone, formaldehyde, glutaraldehyde and paraformaldehyde, the primary antibody is a specific binding antibody of an antigen to be detected, and the fluorescent secondary antibody is a fluorescently-labeled antibody against the primary antibody.
Based on the above technical scheme, preferably, the cell membrane punching solution is a PBS solution containing 2.5% (w/w) sucrose, 20% (v/v) DMSO, 5% (v/v) Tween-20 and 5% (v/v) Triton X-100.
Based on the above technical scheme, preferably, the blocking solution is a sodium azide solution containing 10% (v/v) serum.
On the basis of the technical scheme, preferably, the dehydrating agent comprises a tetrahydrofuran gradient solution.
On the basis of the above technical solution, preferably, the volume ratio of tetrahydrofuran in each gradient of the tetrahydrofuran gradient solution is 50%, 75%, 95% and 100%, respectively.
On the other hand, the invention also provides application of the 3D stereo immunofluorescence staining kit in immunofluorescence staining.
On the basis of the above technical solution, preferably, the application comprises the following steps:
s1, placing the tissue sample in a centrifuge tube, adding a fixative for fixing for more than 24h, discarding the liquid, and washing with PBS buffer solution for 2-3 times, 8h each time;
s2, adding cell membrane punching liquid, incubating in a 37 ℃ gas-heated oscillator for 7-21d for punching, discarding liquid after punching, and washing with PBS buffer solution for 2-3 times, each time for 2 h;
s3, adding a sealing solution, incubating in a 37 ℃ gas heating oscillator for 3d for sealing, discarding the liquid after sealing, and washing with PBS buffer solution for 2-3 times, 2h each time;
s4, adding primary antibodies capable of fully covering the sample, adding 0.1% sodium azide solution for preservative treatment, incubating in a 37 ℃ gas heating oscillator for 14-21d, discarding the liquid after primary antibody incubation is finished, and washing with PBS buffer solution for 2-3 times, 8h each time;
s5, adding a fluorescent secondary antibody diluted by 100 times by PBS buffer solution, adding 0.1% sodium azide solution for preservative treatment, incubating for 7 days in a 37 ℃ gas-heated oscillator, discarding liquid after the secondary antibody incubation is finished, and washing for 2-3 times by PBS buffer solution, wherein each time is 8 hours;
s6, placing the sample after fluorescent labeling in a dehydrating agent, and dehydrating in a dark place at 4 ℃;
s7, performing light-resistant permeation treatment on the dehydrated sample at 4 ℃ by using ion exchange resin;
and S8, embedding the sample by using ion exchange resin, and placing the sample into a 3D scanner for imaging.
On the basis of the technical scheme, preferably, the PBS buffer solution washing is performed by spin washing in a gyrometer.
On the basis of the technical scheme, preferably, the rotation mixing instrument is an American Tiqi digital display rotating speed adjustable test tube mixer.
Based on the above technical solution, in step S2, it is preferable that the liquid is changed every 24 hours during the punching process.
On the basis of the technical scheme, preferably, the permeation treatment specifically comprises the steps of permeating for 3 hours, then replacing a new resin monomer, permeating, then replacing the solution once every 24 hours, and completing the permeation after 72 hours.
Compared with the prior art, the 3D immunofluorescence staining kit and the application thereof have the following beneficial effects:
(1) the invention can carry out fluorescence labeling on the whole large tissue, and finally obtains the 3D digital imaging result and the protein positive data analysis in a video form by scanning through the 3D scanner, compared with the traditional immunofluorescence, the invention can realize absolute quantitative analysis and larger-range protein labeling;
(2) when the paraffin section is manufactured by traditional immunofluorescence staining, tissues are subjected to high-temperature treatment of absolute ethyl alcohol and xylene at the temperature of over 60 ℃, so that the activity of a tissue sample is greatly lost, and the 3D solid immunofluorescence staining can be used for taking local tissue blocks and protecting the activity of the tissues in a mild transparent mode, so that the antibodies and antigens in the tissues are combined to realize protein positioning and marking.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a whole brightfield image of rat brain in example 2.
FIG. 2 is a front view of the fluorescence labeling of rat brain glial cells in example 2.
FIG. 3 is a cross-sectional view of the fluorescence labeling of rat brain glial cells in example 2.
FIG. 4 is a left view of the fluorescent labeling of rat brain glial cells from example 2.
FIG. 5 is a 5-fold enlarged view of the fluorescent marker of rat brain glial cells in example 2.
FIG. 6 is a 10-fold enlarged view of the fluorescent marker of rat brain glial cells in example 2.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
The experimental methods in the following examples, which are not specified under specific conditions, were selected according to the conventional methods and conditions, or according to the commercial instructions. The reagents and starting materials used in the present invention are well known in the art and are readily available on the market.
Example 1
The embodiment provides a 3D immunofluorescence staining kit, which comprises the following reagents:
(1) m PBS buffer.
(2) 0.1% (w/v) sodium azide solution.
(3) Fixing liquid: ethanol, acetone, formaldehyde, glutaraldehyde and paraformaldehyde.
(4) The cell membrane perforating solution is a PBS solution containing 2.5 percent (w/w) of sucrose, 20 percent (v/v) of DMSO, 5 percent (v/v) of Tween-20 and 5 percent (v/v) of Triton X-100, and the preparation method comprises the steps of preparing 0.1M of PBS buffer solution, adding 2.5g of sucrose, 20M of L DMSO, 5M of L Tween-20 and 5M of L of Triton X-100 into the PBS buffer solution, fully stirring and dissolving the mixture, and then using 0.1M of PBS buffer solution to fix the volume to 100M of L to obtain the cell membrane perforating solution.
(5) The preparation method comprises the steps of dissolving 10 mu g of sodium azide in 10m L double distilled water to prepare 0.1% sodium azide solution, and fully mixing and dissolving 9m L0.1.1% sodium azide solution and 1m L serum to obtain the confining liquid.
(6) A first antibody: the antigen to be detected binds specifically to the antibody.
(7) Fluorescent secondary antibody: a fluorescently labeled anti-primary antibody.
(8) Dehydrating agent: tetrahydrofuran gradient solutions, including in particular 50% THF solution, 75% THF solution, 95% THF solution and 100% THF solution (by volume percent).
(9) Ion exchange resin: wokay of national reagents Ltd
15 ion exchange resin (xw90372451)
Example 2 positioning and Observation of rat brain glial cell protein Using 3D stereoimmunofluorescence staining kit
The primary antibody used in this example was specifically GFAP antibody (ab7260) from Abcam, UK, and the secondary antibody was specifically Cy3 fluorescently labeled goat anti-rabbit IgG (ab6939) from Abcam, UK. Before the experiment, whether the expression positioning of the primary antibody is accurate is tested by using a common immunofluorescence method, and if the expression positioning of the primary antibody is accurate, the operation of the embodiment can be carried out. The rotation mixing instrument used in the embodiment is a digital display rotation speed adjustable test tube mixer of the company Jingqi, usa, and the model number of the rotation mixing instrument is TR-02U.
The rat brain tissue glial cell protein localization experiment specifically comprises the following steps:
s1, placing a rat brain block sample in a centrifuge tube, adding 4% paraformaldehyde for fixing for more than 24h, discarding the liquid, adding 0.M PBS buffer solution, placing in a vortex mixer, and carrying out rotary washing for 2-3 times, wherein each time is 8 h;
s2, adding cell membrane punching liquid, incubating in a 37 ℃ gas-heated oscillator for 7-21d for punching, changing liquid once every 24h in the process, discarding liquid after punching, and washing with 0.M PBS buffer solution for 2-3 times, 2h each time;
s3, adding a sealing liquid, incubating in a 37 ℃ gas heating oscillator for 3d for sealing, discarding the liquid after sealing, adding 0.M PBS buffer solution, placing in a vortex mixer, and carrying out rotary washing for 2-3 times, 2h each time;
s4, adding primary antibodies capable of fully covering the sample, adding 0.1% sodium azide solution for preservative treatment, incubating in a 37 ℃ gas heating oscillator for 14-21d, discarding the liquid after primary antibody incubation is finished, adding 0.M PBS buffer solution, placing in a vortex mixer, and carrying out rotary washing for 2-3 times, 8h each time;
s5, adding a fluorescent secondary antibody diluted by 100 times by using 0.M PBS buffer solution, adding 0.1% sodium azide solution for antiseptic treatment, incubating for 7d in a 37 ℃ gas-heated oscillator, discarding the liquid after the secondary antibody incubation is finished, adding 0.M PBS buffer solution, placing in a vortex mixer, and carrying out rotary washing for 2-3 times, wherein each time is 8 hours;
s6, placing the fluorescence-labeled sample in a tetrahydrofuran gradient solution, and performing light-shielding dehydration treatment at 4 ℃, wherein the dehydration treatment is sequentially performed in a 50% THF solution for 3 hours, a 75% THF solution for 3 hours, a 95% THF solution for 3 hours, a 100% THF solution for 3 hours, and then a new 100% THF solution for 5 hours;
s7, performing light-resistant permeation treatment on the dehydrated sample at 4 ℃ by using ion exchange resin, allowing the sample to sink after about 3h of permeation, replacing a new resin monomer for permeation, and replacing the solution once every 24h, wherein the permeation is complete after 72 h;
and S8, embedding the sample by using ion exchange resin, and placing the sample into a 3D scanner for imaging.
The scanning imaging result is shown in fig. 1-6, clear images of rat brain at different visual angles can be observed by using a 3D scanner, 3D three-dimensional protein positioning marking is realized, and protein positive data analysis is performed on the whole body.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (10)
1. A3D immunofluorescence staining kit which characterized in that: the kit comprises a fixing solution, a cell membrane punching solution, a sealing solution, a PBS buffer solution, a primary antibody, a fluorescent secondary antibody, a dehydrating agent and ion exchange resin, wherein the fixing solution is at least one of ethanol, acetone, formaldehyde, glutaraldehyde and paraformaldehyde, the primary antibody is a specific binding antibody of an antigen to be detected, and the fluorescent secondary antibody is a fluorescent-labeled antibody against the primary antibody.
2. The 3D immunofluorescent staining kit according to claim 1, wherein: the cell membrane-perforating solution is a PBS solution containing 2.5% (w/w) sucrose, 20% (v/v) DMSO, 5% (v/v) Tween-20 and 5% (v/v) Triton X-100.
3. The 3D immunofluorescent staining kit according to claim 1, wherein: the confining liquid is sodium azide solution containing 10% (v/v) serum.
4. The 3D immunofluorescent staining kit according to claim 1, wherein: the dehydrating agent comprises a tetrahydrofuran gradient solution.
5. The 3D immunofluorescent staining kit according to claim 4, wherein: the volume ratio of tetrahydrofuran in each gradient of the tetrahydrofuran gradient solution is respectively 50%, 75%, 95% and 100%.
6. The use of the 3D immunofluorescent staining kit according to any one of claims 1 to 5, characterized in that it comprises the following steps:
s1, placing the tissue sample in a centrifuge tube, adding a fixative for fixing for more than 24h, discarding the liquid, and washing with PBS buffer solution for 2-3 times, 8h each time;
s2, adding cell membrane punching liquid, incubating in a 37 ℃ gas-heated oscillator for 7-21d for punching, discarding liquid after punching, and washing with PBS buffer solution for 2-3 times, each time for 2 h;
s3, adding a sealing solution, incubating in a 37 ℃ gas heating oscillator for 3d for sealing, discarding the liquid after sealing, and washing with PBS buffer solution for 2-3 times, 2h each time;
s4, adding primary antibodies capable of fully covering the sample, adding 0.1% sodium azide solution, incubating in a 37 ℃ gas heating oscillator for 14-21d, discarding the liquid after primary antibody incubation is finished, and washing with PBS buffer solution for 2-3 times, wherein each time is 8 h;
s5, adding a fluorescent secondary antibody diluted by 100 times by PBS buffer solution, adding 0.1% sodium azide solution, incubating for 7d in a gas heating oscillator at 37 ℃, discarding the liquid after the secondary antibody incubation is finished, and washing for 2-3 times by PBS buffer solution, wherein each time is 8 hours;
s6, placing the sample after fluorescent labeling in a dehydrating agent, and dehydrating in a dark place at 4 ℃;
s7, performing light-resistant permeation treatment on the dehydrated sample at 4 ℃ by using ion exchange resin;
and S8, embedding the sample by using ion exchange resin, and placing the sample into a 3D scanner for imaging.
7. The use of claim 6, wherein: the PBS buffer washing is performed by spin washing in a gyrometer.
8. The use of claim 7, wherein: the rotation mixing instrument is a test tube mixer with adjustable rotation speed of the American Tiqi digital display.
9. The use of claim 6, wherein: in step S2, the liquid is changed every 24 hours during the punching process.
10. The use of claim 6, wherein: the permeation treatment specifically comprises the steps of permeating for 3 hours, then, replacing a new resin monomer, permeating, then, replacing liquid once every 24 hours, and completing permeation after 72 hours.
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| CN112834737A (en) * | 2020-12-30 | 2021-05-25 | 佳维斯(武汉)生物医药有限公司 | Accurate immunofluorescence labeling method for whole tissue sample |
| CN114705518A (en) * | 2021-12-31 | 2022-07-05 | 武汉巴菲尔生物技术服务有限公司 | Suspension cell incubation staining method and immunofluorescence detection method |
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| CN114705518A (en) * | 2021-12-31 | 2022-07-05 | 武汉巴菲尔生物技术服务有限公司 | Suspension cell incubation staining method and immunofluorescence detection method |
| CN114705518B (en) * | 2021-12-31 | 2024-07-30 | 伊莱瑞特(武汉)生物技术有限公司 | Suspension cell incubation staining method and immunofluorescence detection method |
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