CN110376044B - Neurosphere immunofluorescence staining method - Google Patents
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Abstract
The invention discloses a neurosphere immunofluorescence staining method, which comprises the following steps: (1) cleaning a neurosphere: taking the neurosphere subjected to suspension culture, centrifuging, discarding the supernatant, re-suspending the neurosphere, centrifuging again, and discarding the supernatant; (2) fixing; (3) permeability and sealing; (4) primary antibody incubation; (5) rinsing; (6) incubating a second antibody; (7) rinsing; (8) sealing: adding PBS buffer solution, resuspending, dropping on glass slide, dropping anti-fluorescence quencher, covering with cover glass, pressing neurosphere into flat seal for fluorescent microscope observation. The invention utilizes the characteristics of large diameter and macroscopic view of the neurosphere, directly adopts low-speed centrifugation to collect cells at the bottom of the tube, avoids the loss of the neurosphere in the dyeing process, can keep the original cell shape and internal structure of the neurosphere, and effectively avoids the antigen characteristic change caused in the cell climbing process; the physical extrusion method is adopted to directly carry out tabletting on the nerve ball, the nerve ball is extruded into a film without being sliced, and the requirement on equipment is low.
Description
Technical Field
The invention relates to a neurosphere immunofluorescence staining method, and belongs to the technical field of molecular biology.
Background
Neural stem cells refer to a cell population that has the ability to differentiate into neurons, astrocytes and oligodendrocytes, is capable of self-renewal, and can provide a large number of brain tissue cells. The neural stem cells are obtained and researched by adopting a suspension neurosphere culture method.
The immunofluorescence technique is that a fluorescent pigment which does not affect the activity of antigen-antibody is marked on the antibody (or antigen), and after the fluorescent pigment is combined with the corresponding antigen (or antibody), a specific fluorescence reaction is presented under a fluorescence microscope, so that the tissue or intracellular antigen substance is positioned.
Usually, immunofluorescence staining of neurospheres is to perform cell slide or frozen section on neurospheres to fix the neurospheres on a glass carrier and then perform staining, and the staining steps are complex, the cost is high, and the professional requirements on personnel are high. In addition, the cell slide has the defects that the cells are difficult to adhere to the wall, the antigen characteristics are easy to change, the experimental result is interfered, and the like in the manufacturing process of the cell slide; the frozen section has the defects of high equipment requirement, easy rolling and stripping and the like.
Chinese invention patent CN 102288471 a discloses an immunofluorescence staining method of suspension cells, which is applicable to suspension cells, but has a deficiency in immunofluorescence staining of neurospheres due to the following reasons: firstly, the diameter of the neurosphere is larger, only cells on the surface layer of the neurosphere can be stained when the method is used for staining, and cells inside the neurosphere cannot be stained successfully; secondly, the method is easy to cause cell lysis, and for neurospheres, the original cell form and internal structure are damaged, so that the dyeing result is influenced, and the practical application value is not high. The Chinese invention patent CN 105424450A discloses a suspension cell ball immunofluorescence staining method and a staining device, the method is suitable for suspension cell balls, the immunofluorescence staining of the cell balls by the method needs a special staining device, and is difficult to satisfy in common laboratories; the method is performed in a transparent way and closed respectively, and time is consumed; the confining liquid of the method is goat serum, the confining effect is poor, and non-specificity is easily caused.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides the neurosphere immunofluorescence staining method, which can directly stain neurospheres in culture without performing neurosphere cell slide or frozen section, is not easy to cause cell lysis in the staining process, can well keep the integrity and the antigenic property of the original neurospheres, does not need special devices, saves time, and has good staining effect and strong specificity.
The invention is realized by the following technical scheme:
a method of immunofluorescent staining of neurospheres comprising the steps of:
(1) cleaning neurospheres: placing suspension-cultured neurospheres into a 1.5ml centrifuge tube, centrifuging, discarding the supernatant, adding PBS buffer solution to resuspend the neurospheres, centrifuging again, and discarding the supernatant;
(2) fixing: adding fixative, mixing, standing at room temperature for 30 min, flicking the tube bottom every 5 min to make the neurosphere contact with fixative completely, centrifuging, and discarding the supernatant;
(3) nerve ball penetration and sealing: adding a penetrating and sealing solution, incubating for 30-60 minutes on a shaking table, centrifuging, and removing a supernatant; the penetrating and sealing liquid is formed by mixing 6% BSA solution and 1% Triton X-100 solution in equal volume;
(4) Primary antibody incubation: adding a primary antibody diluted properly, mixing the primary antibody and the primary antibody gently and uniformly, incubating the mixture for 8 to 12 hours at the temperature of 4 ℃, centrifuging the mixture, and removing supernatant;
(5) rinsing: adding PBST (PBS solution containing 0.5% Triton X-100) to resuspend the neurospheres, washing for 10 min by a shaker, centrifuging, and discarding the supernatant; repeated several times, such as 3 times;
(6) and (3) secondary antibody incubation: adding a properly diluted secondary antibody, gently mixing uniformly, incubating for 3 hours in a shaking table, centrifuging, and removing supernatant;
(7) rinsing: adding PBST to resuspend the neurospheres, cleaning for 10 minutes by a shaking table, centrifuging, and discarding the supernatant; repeated several times, such as 3 times;
(8) sealing: adding PBS buffer solution, gently flicking to resuspend the neurospheres, dripping the resuspended neurospheres on a clean glass slide, and placing the slide on a super clean bench to be air-dried in a dark place; dropping a drop of DAPI-containing anti-fluorescence quencher at the neurosphere, covering a cover slip, and gently flattening the neurosphere to seal the neurosphere for observation by a fluorescence microscope.
Further, the neurosphere has a diameter within 200 um.
Further, the specific parameters of the centrifugation are: 400g, centrifuge for 5 minutes.
Further, the fixing agent is 4% paraformaldehyde solution.
Further, the rotational speed of the shaker was 50 rpm.
Further, the specific method for gently mixing the ingredients is as follows: and (3) flicking the tube bottom to suspend the neurosphere, and gently inverting and uniformly mixing for 2-3 times.
Further, the specific way of incubating the antibody is as follows: and (3) diluting the antibody by using a 3% BSA solution according to an antibody specification, and obliquely placing a centrifugal tube to ensure that the neurospheres are fully contacted with the antibody.
Further, the specific way of shaking table washing is: adding PBST into the centrifuge tube, flicking the tube bottom to suspend the neurosphere, gently inverting and mixing for 2-3 times, and then obliquely placing the neurosphere to a shaking table.
The percentages of solutions referred to in the present invention are in the conventional sense, meaning the weight to volume ratio (in g/ml) when the solute is a solid and the volume ratio when the solute is a liquid.
The invention discloses a neurosphere immunofluorescence staining method, which is provided for overcoming the defects in the prior art. The invention adopts a low-speed centrifugation method to collect the tube bottom of the neurosphere, can keep the original cell shape and internal structure of the neurosphere, and effectively avoids the antigen characteristic change caused in the cell climbing process; efficient permeation and sealing are carried out simultaneously under the shaking table oscillation condition, the membrane breaking liquid and the sealing liquid can more effectively contact cells in the neurosphere, and the phenomena of weak dyeing and non-specificity caused by insufficient permeation of the interior of the neurosphere in the conventional method can be effectively overcome; the neurosphere is directly pressed into a film by a physical extrusion method, so that the integrity of the neurosphere is ensured, and the comprehensive analysis of an immunofluorescence staining result is facilitated; need not to carry out the section, require lowly to equipment and personnel, need not special device, general laboratory just can satisfy.
The neurosphere immunofluorescence staining method is simple to operate and low in professional requirements on operators; special equipment and devices are not needed, the basic steps of immunostaining are not changed, and the operation is convenient for operators to master; the antigenic property of the neurosphere can be effectively ensured; the result is special, the cost is saved, and the application value is high.
The various terms and phrases used herein have the ordinary meaning as is well known to those skilled in the art. To the extent that the terms and phrases are not inconsistent with known meanings, the meaning of the present invention will prevail.
Drawings
FIG. 1: human neurosphere nuclear antigen sox2 immunofluorescence staining results are shown in the figure.
Detailed Description
The present invention will be further described with reference to the following examples. However, the scope of the present invention is not limited to the following examples. It will be understood by those skilled in the art that various changes and modifications may be made to the invention without departing from the spirit and scope of the invention.
The present invention has been described generally and/or specifically with respect to materials used in testing and testing methods. Although many materials and methods of operation are known in the art for the purpose of carrying out the invention, the invention is nevertheless described herein in as detail as possible.
Unless otherwise specified, the instruments, reagents, materials and the like used in the following examples are conventional instruments, reagents, materials and the like known in the art and are commercially available. Unless otherwise specified, the experimental methods, detection methods, and the like in the following examples are all conventional experimental methods, detection methods, and the like in the prior art.
Example 1: immunofluorescent staining of neurospheres
The neurosphere is a human neurosphere, the target antigen is a cell nucleus antigen sox2, and the specific steps are as follows:
(1) cleaning a neurosphere: taking 3 neurospheres subjected to suspension culture, wherein the diameter of each neurosphere is 170-200 um, putting the neurospheres into a 1.5ml centrifuge tube, centrifuging 400g for 5 minutes, discarding the supernatant, adding 1ml PBS buffer solution, flicking the bottom of the tube to suspend the neurospheres, gently reversing and mixing for 2-3 times, centrifuging 400g for 5 minutes, and discarding the supernatant.
(2) Fixing: adding 1ml of 4% paraformaldehyde solution, standing at room temperature for 30 minutes, flicking the tube bottom every 5 minutes to make the neurosphere fully contact with the fixing solution, centrifuging 400g for 5 minutes, and discarding the supernatant.
(3) Nerve ball penetration and sealing: permeability and blocking solution 3% BSA (containing 0.5% Triton X-100) was added and incubated on a shaker for 60 minutes at 50 rpm. Then 400g was centrifuged for 5 minutes and the supernatant was discarded.
The permeation and blocking solution is prepared by mixing 6% BSA solution and 1% Triton X-100 solution in equal volume.
(4) Primary anti-incubation: add 300ul of primary antibody diluted in 3% BSA (sox2, rabbit source, 1/200 dilution) and mix gently, then place the tube in a refrigerator at 4 ℃ and incubate overnight. Centrifuge at 400g for 5 min and discard the supernatant.
(5) Rinsing: adding 1ml of PBST (PBS solution containing 0.5% Triton X-100) to flick the tube bottom to suspend the neurospheres, gently inverting and mixing for 2-3 times, then obliquely placing the mixture until the mixture is rinsed for 10 minutes by a shaking table, wherein the rotation speed of the shaking table is 50 revolutions per minute. Centrifuge at 400g for 5 min and discard the supernatant. Repeat 3 times.
(6) And (3) secondary antibody incubation: add 300ul of 3% BSA diluted secondary antibody (goat anti rabbit AF488, 1/1500 diluted) and mix gently, incubate 3 hours on the shaker at 50 rpm. Centrifuge at 400g for 5 min and discard the supernatant.
(7) Rinsing: adding 1ml of PBST (PBS solution containing 0.5% Triton X-100) to flick the tube bottom to suspend the neurospheres, gently inverting and mixing for 2-3 times, then obliquely placing the mixture until the mixture is rinsed for 10 minutes by a shaking table, wherein the rotation speed of the shaking table is 50 revolutions per minute. Centrifuge at 400g for 5 min and discard the supernatant. Repeat 3 times.
(8) Sealing: add 100ul PBS flick to resuspend the neurospheres, drop the resuspended neurospheres onto a clean slide, place in a clean room and air dry in the dark. Dropping a drop of DAPI-containing anti-fluorescence quencher at the neurosphere, covering a cover slip, and gently flattening the neurosphere to seal.
(9) As shown in FIG. 1, the staining of sox2 localized the cell nucleus, and the results are specific as seen in FIG. 1.
The above examples are provided to those of ordinary skill in the art to fully disclose and describe how to make and use the claimed embodiments, and are not intended to limit the scope of the disclosure herein. Modifications apparent to those skilled in the art are intended to be within the scope of the appended claims.
Claims (5)
1. A neurosphere immunofluorescence staining method is characterized by comprising the following steps:
(1) cleaning neurospheres: taking neurospheres which are subjected to suspension culture and have the diameters of 170-200 mu m, centrifuging, discarding the supernatant, adding PBS (phosphate buffer solution) to resuspend the neurospheres, centrifuging again, and discarding the supernatant;
(2) fixing: adding 4% paraformaldehyde solution as fixative, gently mixing, standing at room temperature for 30 min, flicking the tube bottom every 5 min to make the neurosphere fully contact with the fixative, centrifuging, and removing the supernatant;
(3) nerve ball penetration and sealing: adding a transparent and closed solution, and incubating on a shaking table for 30-60 minutes, wherein the rotation speed of the shaking table is 50 revolutions per minute; centrifuging and discarding the supernatant; the penetrating and sealing liquid is formed by mixing 6% BSA solution and 1% Triton X-100 solution in equal volume;
(4) Primary antibody incubation: adding a primary antibody diluted properly, mixing the primary antibody and the primary antibody gently and uniformly, incubating the mixture for 8 to 12 hours at the temperature of 4 ℃, centrifuging the mixture, and removing supernatant;
(5) rinsing: adding PBST (poly-p-phenylenebenzobisthiazole) to resuspend the neurospheres, cleaning by a shaking table, centrifuging, and discarding the supernatant; repeating for several times;
(6) and (3) secondary antibody incubation: adding a properly diluted secondary antibody, gently mixing uniformly, incubating for 3 hours in a shaking table, centrifuging, and removing supernatant;
(7) rinsing: adding PBST (poly-p-phenylenebenzobisthiazole) to resuspend the neurospheres, cleaning by a shaking table, centrifuging, and discarding the supernatant; repeating for several times;
(8) sealing: adding PBS buffer solution, flicking to resuspend the neurospheres, dripping the resuspended neurospheres on a clean glass slide, and placing the glass slide in a super clean bench to be dried in the dark; dripping a drop of anti-fluorescence quencher containing DAPI at the neurosphere, covering a cover glass, gently flattening the neurosphere and sealing the neurosphere for observation by a fluorescence microscope;
the specific parameters of the centrifugation are as follows: 400 g, centrifuge for 5 minutes.
2. The neurosphere immunofluorescent staining method according to claim 1, wherein the specific mode of gentle and uniform mixing is as follows: and (3) flicking the tube bottom to suspend the neurosphere, and gently inverting and uniformly mixing for 2-3 times.
3. The method for immunofluorescent staining of neurospheres according to claim 1, wherein the primary antibody is incubated in a specific manner: and (3) diluting the antibody by using a 3% BSA solution, and obliquely placing a centrifugal tube to ensure that the neurospheres are fully contacted with the antibody.
4. The neurosphere immunofluorescent staining method according to claim 1, wherein the specific way of washing the shaking table is as follows: adding PBST into the centrifuge tube, flicking the tube bottom to suspend the neurosphere, gently inverting and mixing for 2-3 times, and then obliquely placing the neurosphere to a shaking table.
5. The method of neurosphere immunofluorescent staining according to claim 1 or 4, wherein: the PBST was PBS containing 0.5% Triton X-100.
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