CN110923141A - Improved six-hole plate and cell immunofluorescence staining method based on same - Google Patents
Improved six-hole plate and cell immunofluorescence staining method based on same Download PDFInfo
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Abstract
The invention discloses an improved six-hole plate and a cell immunofluorescence staining method based on the same, and belongs to the technical field of biomedical equipment. According to the invention, the reserved groove is arranged at the side edge of the inverted circular truncated cone-shaped groove, so that a microscopic cover glass can be conveniently taken and placed by using tweezers during an immunofluorescence experiment, the operation time of an experimenter is saved, and the working efficiency of the experimenter is improved; the method can improve the quality of immunofluorescence staining, reduce the time spent in antibody incubation, avoid the microscopic cover glass from being cracked in the taking and placing process, reduce the using amount of the antibody and save the cost.
Description
Technical Field
The invention relates to the technical field of biomedical equipment, in particular to an improved six-hole plate and a cell immunofluorescence staining method based on the six-hole plate.
Background
Immunofluorescence is an experimental method commonly used in cell and molecular biology. The technique is to detect a corresponding antigen (antibody) in a tissue or a cell by using a fluorescent antibody (antigen) labeled with fluorescein as a probe. Fluorescein is deposited on the antigen-antibody complex formed in the tissue or cell, the sample is observed under a fluorescence microscope, the fluorescein emits fluorescence under the irradiation of exciting light, the antigen or antibody is positioned through the cell or tissue where the fluorescence is located, and meanwhile, the sample can be quantified by utilizing a quantification technology. Fluorescein can label both antibody and antigen, but because the structure and physicochemical properties of the antigen are complex, the condition of fluorescein labeling is not easy to control, and the antibody is usually labeled by fluorescein, so the technology is also called as fluorescent antibody technology.
Immunofluorescence assays are classified into indirect immunofluorescence assays and direct immunofluorescence assays, according to the use of antibodies. The indirect immunofluorescence analysis method labels an antigen through an antigen-specific first antibody (primary antibody) and then specifically labels the primary antibody through a second antibody (secondary antibody) coupled with a fluorescent molecule, so that the site where the antigen is located emits fluorescence. Direct immunofluorescence analysis is a method of directly labeling an antigen with an antigen-specific antibody coupled to a fluorescent molecule. Either indirect or direct immunofluorescence analysis, cell slide-based immunofluorescence methods are applicable.
At present, some detection methods and products or related tools for cell culture are researched and developed mainly on the basis of an immunofluorescence technology in China, and related patent applications such as a glass bottom culture plate convenient for preparation of an immunofluorescence sample disclosed in a Chinese utility model patent with the patent application number of CN201821041666.1 and an immunofluorescence kit for detecting different sites of HER2 antigen disclosed in a Chinese invention patent with the patent application number of CN201810908228.9 and application thereof. These methods and devices are too complex to operate and have a deep fluorescent background.
The traditional cell immunofluorescence staining method mainly comprises the following steps:
1. cells were seeded into a common six-well plate plated with microscopic coverslips. 2. And (4) preparing cell slide when the cell grows to reach the fullness of about 70 percent of the area of the microscopic cover glass. 3. And absorbing and removing the culture medium, washing the microscopic cover glass with PBS (phosphate buffer solution) for 2 times, adding 4% paraformaldehyde solution into each culture well, fixing the cells for 30 minutes at room temperature, absorbing and removing the fixing solution in the six-well plate, washing the microscopic cover glass with the PBS solution for 3 times, and washing off redundant paraformaldehyde after 5 minutes each time. 4. 0.5% of the transmembrane solution (500. mu.L of Triton X-100 in 100mL of PBS) was added to each culture well and incubated at room temperature for 30 minutes; and after the membrane penetration is finished, sucking and discarding 0.5 percent of membrane penetration liquid. 5. Cells were blocked with 5% BSA for 1 hour. 6. Rabbit-derived γ -H2AX primary antibodies were raised against 1: diluting by a multiple of 1000, dripping the diluted antibody on a plastic plate paved with a sealing film, clamping a glass slide with a microscope cover out of a six-hole plate by using forceps, enabling one surface containing cells to be in contact with the antibody diluent, reversely buckling the glass slide on the sealing film, and incubating the antibody and the cells for 1.5 hours at room temperature. 7. After the 5% BSA was aspirated, the microscope coverslip was clamped back to the six well plate with forceps, after which the microscope coverslip was washed 3 times 5 minutes each time with 0.1% PBST solution on a shaker. 8. Alexa488 fluorescently labeled goat anti-rabbit secondary antibody was added to 0.1% PBST solution at a ratio of 1: diluting by a multiple of 1000, dripping the diluted antibody on a plastic plate paved with a sealing film, clamping the glass microscope cover sheet out of a six-hole plate by using tweezers, enabling one surface containing cells to be in contact with the antibody diluent, reversely buckling the glass microscope cover sheet on the sealing film, incubating the glass microscope cover sheet at room temperature in a dark place for 1 hour, clamping the glass microscope cover sheet back into the six-hole plate by using the tweezers, and then washing the glass microscope cover sheet on a shaking table in the dark place for 3 times by using 0.1% PBST solution for 5 minutes each time. 9. Nuclei were stained with 1. mu.g/mL Hoechst33342 in the dark for 8 minutes, and washed 3 times with PBS solution in the dark for 5 minutes each. 11. Dropping a drop of the anti-fluorescence quenching blocking tablet on the glass slide, and reversely covering the side with the cells in the antibody-labeled microscopic cover glass on the anti-fluorescence quenching blocking tablet, and storing at-20 ℃.
As a basic container for a culture medium, a six-well plate is widely used not only for general cell culture but also for immunofluorescent-stained cell slide. The prior six-hole plate generally comprises a six-hole plate box body, a cover and culture holes arranged on the six-hole plate box body.
In the cell climbing process of immunofluorescence staining, often put into micro cover glass in six orifice plates and carry out the cell climbing after, add the complete culture medium of DMEM, the cultivation of carrying out the cell on micro cover glass, must press from both sides out micro cover glass with tweezers after the cultivation is accomplished, carry out the incubation of primary antibody and two antibodies, but ordinary six orifice plates are because the bottom is flat, micro cover glass is flat, lead to micro cover glass tightly to laminate in six orifice plates round hole bottom plane, difficult taking out, the condition of piece appears even, cause unnecessary influence to the experimental result.
When primary antibody and secondary antibody incubation is carried out, the microscopic cover glass is frequently taken out and put back, time and energy are wasted, the defects of the traditional immunofluorescence staining method are obvious, a large amount of time is needed in the experiment steps, the microscopic cover glass is easy to break, and experiment failure is easily caused. Therefore, an improved six-well plate and a cell immunofluorescence staining method based on the six-well plate are provided.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: on the premise of improving the quality of immunofluorescence staining, the time spent on antibody incubation is reduced, the using amount of the antibody is reduced, and the phenomenon of fragmentation in the process of taking and placing microscopic cover slips is avoided.
The technical scheme includes that the culture plate comprises a culture plate body and six culture holes, openings of the six culture holes are upward and are distributed on the culture plate body in an array mode, an inverted-truncated-cone-shaped groove with an upward opening is formed in the bottom end surface of each culture hole, the diameter of a microscopic cover glass is larger than the diameter of the bottom end surface of the inverted-truncated-cone-shaped groove and smaller than the diameter of an opening at the upper end of the inverted-truncated-cone-shaped groove, a reserved groove for inserting tweezers is formed in the side edge of the inverted-truncated-cone-shaped groove, the microscopic cover glass is placed inside the inverted-truncated-cone-shaped groove during cell culture, and the microscopic cover glass can be clamped up by inserting the tweezers into the reserved groove during immunofluorescence experiments after the culture is finished. Through set up inverted circular truncated cone shape recess in the bottom of cultivateing the hole, can conveniently put into wherein with microscopic cover glass, and the bottom surface of microscopic cover glass can not with the bottom surface direct contact of inverted circular truncated cone shape recess, reduced the contact force between microscopic cover glass and the cultivation hole bottom surface, avoided the trouble that is difficult to take out when microscopic cover glass directly pastes with cultivation hole bottom surface, also avoided frequently taking out to put back and probably to lead to the cracked condition of microscopic cover glass to take place.
Furthermore, the six culture holes are distributed on the culture plate body in an array of two rows and three columns, and the distances between every two adjacent culture holes are equal. The equal-distance uniform arrangement can increase the beauty of the six-hole plate to a certain extent.
Furthermore, the six-hole plate further comprises a cover body, wherein the cover body is arranged at the upper end of the culture plate body, and the upper part of the culture plate body can be wrapped and covered by the cover body, so that the culture plate body is sealed.
Furthermore, the preformed groove is a triangular conical groove, and the triangular conical groove extends downwards along the side edge of the inverted frustum-shaped groove until the bottom end surface of the inverted frustum-shaped groove is stopped. Set up the reservation groove through the side edge at inverted circular truncated cone shape recess, in carrying out the immunofluorescence experiment process, can conveniently get through tweezers to get when the cell on microscopic cover glass is hatched the antibody and put microscopic cover glass, add the antibody diluent in inverted circular truncated cone shape recess, make the one side that microscopic cover glass contains the cell contact with the antibody diluent, the back-off is in inverted circular truncated cone shape recess. After incubation is finished, the cell-containing surface of the microscopic cover glass is placed upwards in the inverted truncated cone-shaped groove, so that the operation time of experimenters is saved, and the working efficiency of the experimenters is improved.
Furthermore, the preformed groove is a semi-circular conical groove, and the semi-circular conical groove extends downwards along the side edge of the inverted frustum-shaped groove until the bottom end surface of the inverted frustum-shaped groove is cut off.
Furthermore, the improved six-hole plate further comprises a bottom plate and a circular sealing cover, wherein the circular sealing cover is arranged at the bottom end of each inverted truncated cone-shaped groove and seals the bottom end of each inverted truncated cone-shaped groove, and the bottom plate is inserted into the lower end of the culture plate body, is positioned at the lower end of the circular sealing cover and upwards extrudes the circular sealing cover.
Furthermore, a serrated groove is arranged between the circular sealing cover and the culture plate body, the serrated groove is respectively arranged on the circular sealing cover and the culture plate body, the serrated groove on the circular sealing cover is arranged on the edge of the upper surface of the circular sealing cover, and the serrated groove on the culture plate body is arranged on the surface of the joint of the culture plate body and the circular sealing cover.
The invention also provides a cell immunofluorescence staining method based on the improved six-hole plate, which comprises the following steps:
s1: cell climbing sheet
The microscopic coverslips were placed into the inverted frustum-shaped recess with tweezers one by one in the clean bench, after which 1.2X 10 were placed5Planting Hep G2 cells in the modified six-well plate containing the microscopic cover glass in the inverted truncated cone-shaped groove, and allowing the cells to grow on the microscopic cover glass for 24 hours; the diameter of the microscopic cover glass is larger than the diameter of the bottom end surface of the inverted circular truncated cone-shaped groove and smaller than the diameter of the opening at the upper end of the inverted circular truncated cone-shaped groove, so that the microscopic cover glass can be conveniently placed into the inverted circular truncated cone-shaped groove, a reserved groove for inserting tweezers is formed in the side edge of the inverted circular truncated cone-shaped groove, the microscopic cover glass is placed inside the inverted circular truncated cone-shaped groove when cell culture is carried out, and the microscopic cover glass can be clamped by inserting the tweezers into the reserved groove. The bottom end of the culture hole is provided with the inverted frustum-shaped groove, so that the microscopic cover glass can be conveniently placed in the groove;
s2: toxin treatment
Continuously culturing Hep G2 cells in the improved six-hole plate for 24 hours, after the cells grow to a logarithmic growth phase, removing the original culture medium, culturing the cells for 24 hours by using a DMEM complete culture medium containing microcystin-LR (MC-LR) with different concentrations, and adding an isovolumetric DMEM complete culture medium into a control group; when preparing MC-LR, firstly, 3 centrifuge tubes with 5mL are taken, corresponding to three concentrations of 1 μ M, 0.1 μ M and 0.01 μ M respectively, 2450 μ L, 2250 μ L of DMEM complete culture medium is added into the three centrifuge tubes respectively, 50 μ L of pure MC-LR stock solution is added into the centrifuge tube of a 1 μ M treatment group containing 2450 μ L of DMEM complete culture medium, and the blowing and sucking are uniform; then 250 mul of the mixed solution is taken from the centrifuge tube of the 1 mul processing group and added into the centrifuge tube of the 0.1 mul processing group containing 2250 mul of DMEM complete medium, and the mixture is evenly blown and sucked; then 250 mul of mixed solution is taken from the centrifuge tube of the 0.1 mul processing group and added into the centrifuge tube of the 0.01 mul processing group, and the blowing and the suction are uniform; finally, 2250. mu.L of 1. mu.M, 2250. mu.L of 0.1. mu.M and 2500. mu.L of 0.01. mu.M MC-LR working solutions were prepared, 2mL of each MC-LR working solution with different concentrations was added to the corresponding wells, and toxin-treated for 24 hours;
s3: cell fixation
Removing a toxin-containing DMEM complete culture medium by suction, cleaning the microscopic cover glass with a PBS solution for 2 times, adding 1mL of a 3% glutaraldehyde solution into each culture well, fixing cells for 10 minutes at room temperature, removing a fixing solution in the improved six-well plate by suction, cleaning the microscopic cover glass with the PBS solution for 3 times, wherein the cleaning time is 5 minutes each time, and washing off residual glutaraldehyde solution;
s4: penetrating film
1mL of 1% transmembrane solution (1mL of Triton X-100 in 100mL of PBS) was added to each culture well and incubated at room temperature for 10 minutes; after membrane penetration is finished, 1% of membrane penetrating liquid is sucked and discarded, and the membrane is washed for 1 time by PBS solution;
s5: sealing of
Adding 1mL of protein-free rapid blocking solution (1X) to each culture well, and blocking for 10 minutes;
s6: primary antibody incubation
Recovering the protein-free rapid blocking solution (1X), and subjecting the rabbit-derived γ -H2AX primary antibody to a reaction in the presence of the protein-free rapid blocking solution (1X) according to a ratio of 1: diluting by a multiple of 1000, adding an antibody diluent into the inverted-truncated-cone-shaped groove, stretching into the inverted-truncated-cone-shaped groove along the reserved groove by using a pair of tweezers to clamp the microscopic cover slip, enabling one surface containing cells to be in contact with the antibody diluent, reversely buckling the groove in the inverted-truncated-cone-shaped groove, incubating for 30 minutes at 37 ℃, stretching into the inverted-truncated-cone-shaped groove along the reserved groove by using the pair of tweezers to clamp the microscopic cover slip, enabling one surface containing cells to face upwards, sucking and discarding the primary antibody diluent in the inverted-truncated-cone-shaped groove by using a pipette, and cleaning the microscopic cover slip for 6 times by using 0.2% PBST solution (200 mu L Tween-20 dissolved in 100mL PBS), wherein the cleaning time is 5 minutes each time so as to remove residual primary antibody;
s7: incubation with secondary antibody
Alexa488 fluorescently labeled goat anti-rabbit secondary antibody was added to protein-free rapid blocking solution (1 ×) according to 1: diluting by a multiple of 1000, adding an antibody diluent into the inverted-truncated-cone-shaped groove, stretching into the inverted-truncated-cone-shaped groove along the reserved groove by using a pair of tweezers to clamp the microscopic cover glass, enabling one surface containing cells to be in contact with the antibody diluent, reversely buckling the inverted-truncated-cone-shaped groove, incubating at 37 ℃ in a dark place for 20 minutes, stretching into the inverted-truncated-cone-shaped groove along the reserved groove by using the pair of tweezers to clamp the microscopic cover glass, enabling one surface containing cells to face upwards, sucking and discarding the secondary antibody diluent in the inverted-truncated-cone-shaped groove by using a pipettor, and cleaning the microscopic cover glass 6 times in the dark place by using 0.2% PBST solution, wherein the cleaning time is 5 minutes each time so as to remove residual;
s8: nuclear counterstaining
Staining the nucleus with 1 mu g/mL Hoechst33342 in the dark for 2 minutes, and washing 5 times in the dark with PBS solution, wherein the washing time is 5 minutes each time;
s9: color development
Dropping an anti-fluorescence quenching blocking piece on a glass slide, then reversely buckling one side of the antibody-labeled microscopic cover glass, which is provided with cells, on the anti-fluorescence quenching blocking piece, sucking away the redundant anti-fluorescence quenching blocking piece around the microscopic cover glass, and then observing fluorescence under a fluorescence microscope and taking a picture.
Furthermore, in step S1, before cell slide, the microscopic glass slide is placed in a culture dish with a diameter of 100mm, ethanol with a purity of 75% is poured into the culture dish, the culture dish is slowly shaken for 24 hours on a shaking table, then placed in ultraviolet rays for 30 minutes, finally washed for 2 times by the basic culture medium, then placed in the culture dish with a diameter of 100mm containing the basic culture medium, sealed for the periphery by a sealing film, and then placed in an environment at 4 ℃ for soaking for more than 6 hours for storage and standby.
Further, in the steps S6 and S7, the dilution of the antibody is added in an amount of 60 μ L during both the primary antibody incubation and the secondary antibody incubation.
Compared with the prior art, the invention has the following advantages:
according to the six-hole plate, the inverted circular truncated cone-shaped groove is formed in the bottom end of the culture hole, so that a microscopic cover glass can be conveniently placed in the six-hole plate, the bottom surface of the microscopic cover glass cannot be directly contacted with the bottom surface of the inverted circular truncated cone-shaped groove, the contact force between the microscopic cover glass and the bottom surface of the culture hole is reduced, the trouble that the microscopic cover glass is difficult to take out when the microscopic cover glass is directly attached to the bottom surface of the culture hole is avoided, and the situation that the microscopic cover glass is broken due to frequent taking out and putting back is also avoided; set up the reservation groove through the side reason at inverted circular truncated cone shape recess, when carrying out the immunofluorescence experiment, can conveniently get through tweezers and put microscopic cover glass, saved experimenter's operating time to make experimenter's work efficiency can improve.
The immunofluorescence staining method is simple and convenient to operate and easy to master, and reduces the overall time of an immunofluorescence experiment; the improved six-hole plate obviously reduces the usage amount of primary antibodies and secondary antibodies, thereby saving the cost; the fixed penetration effect of the used 3% glutaraldehyde stationary liquid on cells is better; the protein-free rapid sealing liquid has good sealing effect and high sealing speed, is particularly suitable for sealing phosphorylated antigens, saves time and reduces the background of a fluorescent picture; the antibody is diluted by the recovered protein-free rapid blocking solution (1X), so that the nonspecific binding of the antibody can be reduced, and the cost of using the protein-free rapid blocking solution (1X) is saved; compared with the PBST solution with the concentration of 0.1%, the PBST solution with the concentration of 0.2% has better cleaning effect on residual primary antibody and secondary antibody; compared with the membrane penetration solution with the concentration of 0.5%, the membrane penetration solution with the concentration of 1% has better effect of increasing the permeability of cell membranes.
Drawings
FIG. 1 is a top view of a culture plate body according to a first embodiment of the present invention;
FIG. 2 is a schematic view of a combination of an inverted truncated cone-shaped groove and a triangular pyramid-shaped groove according to an embodiment of the present invention;
FIG. 3 is a schematic view of the combination of the base plate and the circular cover according to one embodiment of the present invention;
FIG. 4 is a bottom view of the improved six-hole plate according to the first embodiment of the present invention;
FIG. 5 is a partial schematic view of a bottom plate inserted into a channel strip in accordance with an embodiment of the present invention;
FIG. 6 is a partial schematic view of a circular cover pressed against a culture plate according to an embodiment of the present invention;
FIG. 7 is a schematic diagram of the general flow of the cellular immunofluorescence staining method based on the improved six-well plate of the present invention.
FIG. 8 is a graph of staining of cell nuclei using a conventional immunofluorescence staining method using Hoechst33342 dye;
FIG. 9 is a graph of staining of gamma-H2 AX lesion spots in cell nuclei using a conventional immunofluorescent staining method with rabbit-derived gamma-H2 AX primary antibody and Alexa488 fluorescently labeled goat anti-rabbit secondary antibody;
FIG. 10 is a composite view of FIGS. 8 and 9;
FIG. 11 is a graph showing the staining of nuclei with Hoechst33342 dye in one embodiment of the present invention;
FIG. 12 is a graph showing the staining of gamma-H2 AX lesion spots in nuclei using rabbit-derived gamma-H2 AX primary antibody and Alexa488 fluorescently labeled goat anti-rabbit secondary antibody in accordance with the first embodiment of the present invention;
fig. 13 is a composite view of fig. 11 and 12.
In FIGS. 1 to 6: 1. culturing the plate body; 2. rounding the frustum-shaped groove; 3. a triangular conical groove; 4. a culture well; 5. a circular cover; 51. a saw-toothed groove; 6. groove strips; 7. a base plate; 8. digging a table.
Detailed Description
The following examples are given for the detailed implementation and specific operation of the present invention, but the scope of the present invention is not limited to the following examples.
Example one
As shown in fig. 1 to 7, the present embodiment provides the following technical solutions: six orifice plates of improvement, including culture plate body 1 and six culture holes 4, six culture holes 4's opening all upwards, and its array distribution is in culture plate body 1 is last, culture holes 4's bottom is seted up opening ascending inverted circular truncated cone recess 2 on the surface, and the diameter of microscopic cover glass is greater than inverted circular truncated cone recess 2's bottom surface diameter is less than inverted circular truncated cone recess 2's upper end opening diameter, microscopic cover glass just can conveniently put into inverted circular truncated cone recess 2 like this, the reserve tank that is used for inserting tweezers is offered to inverted circular truncated cone recess 2's side reason, and when carrying out cell culture, microscopic cover glass arranges in inverted circular truncated cone recess 2's inside is through inserting tweezers can press from both sides microscopic cover glass in the reserve tank. Through set up inverted circular truncated cone shape recess 2 in the bottom of cultivateing hole 4, can conveniently put into it with microscopic cover glass, and the bottom surface of microscopic cover glass can not laminate with the bottom surface direct contact of inverted circular truncated cone shape recess 2, reduced the contact force between microscopic cover glass and the cultivation hole 4, avoided the trouble that is difficult to take out when microscopic cover glass directly laminates with cultivateing hole 4 bottom surface, also avoided frequently taking out to put back and probably lead to the cracked condition of microscopic cover glass to take place.
Six culture holes 4 are distributed on the culture plate body 1 according to two rows and three columns, and the distance between two adjacent culture holes 4 is equal. The equal-distance uniform arrangement can increase the beauty of the six-hole plate to a certain extent.
The six-hole plate further comprises a cover body, the cover body is arranged at the upper end of the culture plate body 1, and the upper portion of the culture plate body 1 can be wrapped and covered by the cover body, so that the culture plate body 1 is sealed.
In this embodiment the preformed groove is a triangular pyramid-shaped groove 3, and the triangular pyramid-shaped groove 3 extends downwards along the side edge of the reverse circular truncated cone-shaped groove 2 to the bottom end surface of the reverse circular truncated cone-shaped groove 2.
Six orifice plates of improvement still include bottom plate 7, circular closing cap 5 sets up in the bottom of each inverted circular truncated cone shape recess 2 and seals its bottom, bottom plate 7 is pegged graft cultivate the lower extreme of plate body 1, the lower extreme both sides edge and the back edge of cultivating plate body 1 all are equipped with channel bar 6, channel bar 6 and cultivation plate body 1 integrated into one piece, the inside of channel bar 6 is equipped with horizontal slot, just insert inside horizontal slot at the edge of bottom plate 7, the front end middle part position of bottom plate 7 is equipped with the platform 8 of digging of buckling downwards, be used for the convenience with 7 dismouting of bottom plate.
The bottom plate 7 is positioned at the lower end of the circular sealing cover 5, the integrally formed bosses are arranged at the positions, close to the circular sealing covers 5, of the upper surface of the bottom plate 7, and the connecting parts between the bosses and the bottom plate 7 are in smooth curved surface transition, so that the circular sealing covers 5 can be upwards extruded when the bottom plate 7 is inserted, and are tightly pressed.
After the six-hole plate is used, the bottom plate 7 can be drawn out, then the circular sealing covers 5 below the culture holes 4 are taken out, the inside of the inverted circular truncated cone-shaped groove 2 can be conveniently cleaned, and the cleaning is more thorough. And then, after acid soaking treatment, drying and ultraviolet sterilization, the six-hole plate can be repeatedly used in some basic experiments with low requirements after being assembled, so that certain cost can be saved.
The circular closing cap 5 with be provided with zigzag groove 51 between the culture plate body 1, zigzag groove 51 sets up respectively circular closing cap 5 with on the culture plate body 1, on the circular closing cap 5 zigzag groove 51 sets up at circular closing cap 5 upper end surface edge, on the culture plate body 1 zigzag groove 51 sets up culture plate body 1 with circular closing cap 5 junction surface has placed a sealed cushion on the face of meeting of two zigzag grooves 51, can regularly change according to the service behavior of sealed cushion.
The embodiment also provides a cell immunofluorescence staining method, which comprises the following steps:
s1: cell climbing sheet
The microscopic coverslips were placed into the inverted frustum-shaped recess with tweezers one by one in the clean bench, after which 1.2X 10 were placed5Planting Hep G2 cells in the modified six-well plate containing the microscopic cover glass in the inverted truncated cone-shaped groove, and allowing the cells to grow on the microscopic cover glass for 24 hours; since the diameter of the microscopic cover glass is larger than the diameter of the bottom end surface of the reverse circular truncated cone-shaped groove and smaller than the diameter of the reverse circular truncated cone-shaped grooveThe diameter of the opening at the upper end is such that the microscopic cover glass can be conveniently placed in the inverted frustum-shaped groove, the triangular conical groove for inserting the tweezers is formed in the side edge of the inverted frustum-shaped groove, the microscopic cover glass is placed in the inverted frustum-shaped groove when cell culture is carried out, and the microscopic cover glass can be clamped up by inserting the tweezers into the triangular conical groove. The bottom end of the culture hole is provided with the inverted frustum-shaped groove, so that the microscopic cover glass can be conveniently placed in the groove;
s2: toxin treatment
Continuously culturing Hep G2 cells in the improved six-hole plate for 24 hours, after the cells grow to a logarithmic growth phase, removing the original culture medium, culturing the cells for 24 hours by using DMEM complete culture medium containing MC-LR with different concentrations, and adding the DMEM complete culture medium with the same volume to a control group; when preparing MC-LR, firstly, 3 centrifuge tubes with 5mL are taken, corresponding to three concentrations of 1 μ M, 0.1 μ M and 0.01 μ M respectively, 2450 μ L, 2250 μ L of DMEM complete culture medium is added into the three centrifuge tubes respectively, 50 μ L of pure MC-LR stock solution is added into the centrifuge tube of a 1 μ M treatment group containing 2450 μ L of DMEM complete culture medium, and the blowing and sucking are uniform; then 250 mul of the mixed solution is taken from the centrifuge tube of the 1 mul processing group and added into the centrifuge tube of the 0.1 mul processing group containing 2250 mul of DMEM complete medium, and the mixture is evenly blown and sucked; then 250 mul of mixed solution is taken from the centrifuge tube of the 0.1 mul processing group and added into the centrifuge tube of the 0.01 mul processing group, and the blowing and the suction are uniform; finally, 2250. mu.L of 1. mu.M, 2250. mu.L of 0.1. mu.M and 2500. mu.L of 0.01. mu.M MC-LR working solutions were prepared, 2mL of each MC-LR working solution with different concentrations was added to the corresponding wells, and toxin-treated for 24 hours;
s3: cell fixation
Removing a toxin-containing culture medium by suction, cleaning the microscopic cover glass with a PBS (phosphate buffer solution) solution for 2 times, adding 1mL of a 3% glutaraldehyde solution into each culture well, fixing cells for 10 minutes at room temperature, removing a fixing solution in the improved six-well plate by suction, cleaning the microscopic cover glass with the PBS solution for 3 times, wherein the cleaning time is 5 minutes each time, and washing away residual glutaraldehyde;
s4: penetrating film
Adding 1mL of 1% transmembrane solution into each culture well, and incubating for 10 minutes at room temperature; after membrane penetration is finished, 1% of membrane penetrating liquid is sucked and discarded, and the membrane is washed for 1 time by PBS solution;
s5: sealing of
Adding 1mL of protein-free rapid blocking solution (1X) to each culture well, and blocking for 10 minutes;
s6: primary antibody incubation
Recovering the protein-free rapid blocking solution (1X), and subjecting the rabbit-derived γ -H2AX primary antibody to a reaction in the presence of the protein-free rapid blocking solution (1X) according to a ratio of 1: diluting by a multiple of 1000, adding an antibody diluent into the inverted frustum-shaped groove, stretching the antibody diluent into the inverted frustum-shaped groove along the triangular conical groove by using a pair of tweezers to clamp the microscopic cover glass, enabling one surface containing cells to be in contact with the antibody diluent, reversely buckling the antibody diluent in the inverted frustum-shaped groove, incubating at 37 ℃ for 30 minutes, stretching the antibody diluent into the inverted frustum-shaped groove along the triangular conical groove by using the pair of tweezers to clamp the microscopic cover glass, enabling one surface containing cells to face upwards, sucking and discarding the primary antibody diluent in the inverted frustum-shaped groove by using a pipettor, and cleaning the microscopic cover glass 6 times by using 0.2% PBST solution, wherein the cleaning time is 5 minutes each time so as to remove the residual primary antibody;
s7: incubation with secondary antibody
Alexa488 fluorescently labeled goat anti-rabbit secondary antibody was added to protein-free rapid blocking solution (1 ×) according to 1: diluting by a multiple of 1000, adding an antibody diluent into the inverted frustum-shaped groove, stretching the antibody diluent into the inverted frustum-shaped groove along the triangular conical groove by using a pair of tweezers to clamp the microscopic cover glass, enabling one surface containing cells to be in contact with the antibody diluent, reversely buckling the antibody diluent in the inverted frustum-shaped groove, incubating the antibody diluent in a dark place at 37 ℃ for 20 minutes, stretching the antibody diluent into the inverted frustum-shaped groove along the triangular conical groove by using the pair of tweezers to clamp the microscopic cover glass, enabling one surface containing cells to face upwards, sucking and discarding the primary anti-diluent in the inverted frustum-shaped groove by using a pipettor, and cleaning the microscopic cover glass 6 times in a dark place by using 0.2% PBST solution, wherein the cleaning time is 5 minutes each time so as to remove residual secondary antibodies;
s8: nuclear counterstaining
Staining the nucleus with 1 mu g/mL Hoechst33342 in the dark for 2 minutes, and washing 5 times in the dark with PBS solution, wherein the washing time is 5 minutes each time;
s9: color development
Dropping an anti-fluorescence quenching blocking piece on a glass slide, then reversely buckling one surface of the antibody-marked microscopic cover glass, which is provided with cells, on the anti-fluorescence quenching blocking piece, sucking and removing the redundant anti-fluorescence quenching blocking piece around the microscopic cover glass, and then observing fluorescence under a fluorescence microscope and taking a picture.
In step S1, before cell climbing, the microscopic glass slide is placed in a culture dish with a diameter of 100mm, ethanol with a purity of 75% is poured into the culture dish, the microscopic glass slide is slowly shaken for 24 hours on a shaking table, then placed in ultraviolet rays for irradiation for 30 minutes, finally washed for 2 times by a basic culture medium and then placed in the culture dish with a diameter of 100mm containing the basic culture medium, the periphery of the culture dish is sealed by a sealing film, and the culture dish is placed in a refrigerator with a temperature of 4 ℃ for soaking for more than 6 hours and stored for later use.
In steps S6 and S7, the antibody dilution was added in an amount of 60 μ L during both primary antibody incubation and secondary antibody incubation.
Example two
The embodiment provides the technical scheme that: six orifice plates of improvement, including cultivateing plate body and six cultivation holes, six the opening of cultivateing the hole all upwards, and its array distribution is in cultivate on the plate body, cultivate the bottom of hole and seted up the ascending reverse circular truncated cone recess of opening on the surface, the diameter of micro-cover glass slightly is greater than the bottom surface diameter of reverse circular truncated cone recess is less than the upper end opening diameter of reverse circular truncated cone recess, the reserve tank that is used for inserting tweezers is offered to the side reason of reverse circular truncated cone recess, and when carrying out cell culture, micro-cover glass arranges in the inside of reverse circular truncated cone recess, through inserting tweezers can press from both sides micro-cover glass in the reserve tank. Through set up inverted circular truncated cone shape recess in the bottom of cultivateing the hole, can conveniently put into wherein with microscopic cover glass, and the bottom surface of microscopic cover glass can not laminate with the bottom surface direct contact of inverted circular truncated cone shape recess, reduced the contact force between microscopic cover glass and the cultivation hole bottom surface, avoided the trouble that is difficult to take out when microscopic cover glass directly laminates with cultivation hole bottom surface, also avoided frequently taking out to put back and probably to lead to the cracked condition of microscopic cover glass to take place.
Six culture holes are distributed on the culture plate body according to two rows and three columns, and the distance between every two adjacent culture holes is equal. The equal-distance uniform arrangement can increase the beauty of the six-hole plate to a certain extent.
The six-hole plate further comprises a cover body, the cover body is arranged at the upper end of the culture plate body, and the upper portion of the culture plate body can be wrapped and covered by the cover body, so that the culture plate body is sealed.
In this embodiment, the preformed groove is a semi-circular conical groove, and the semi-circular conical groove extends downwards along the side edge of the inverted frustum-shaped groove and is stopped at the bottom end surface of the inverted frustum-shaped groove. Set up half circular cone shape recess through the side reason in radius platform concave groove, when carrying out the immunofluorescence experiment, can conveniently get through tweezers and put microscopic cover glass, saved experimenter's operating time to make experimenter's work efficiency can improve.
Six orifice plates of improvement still include bottom plate 7, circular closing cap 5 sets up in the bottom of each inverted circular truncated cone shape recess 2 and seals its bottom, bottom plate 7 is pegged graft cultivate the lower extreme of plate body 1, the lower extreme both sides edge and the back edge of cultivating plate body 1 all are equipped with channel bar 6, channel bar 6 and cultivation plate body 1 integrated into one piece, the inside of channel bar 6 is equipped with horizontal slot, just insert inside horizontal slot at the edge of bottom plate 7, the front end middle part position of bottom plate 7 is equipped with the platform 8 of digging of buckling downwards, be used for the convenience with 7 dismouting of bottom plate.
The bottom plate 7 is positioned at the lower end of the circular sealing cover 5, the integrally formed bosses are arranged at the positions, close to the circular sealing covers 5, of the upper surface of the bottom plate 7, and the connecting parts between the bosses and the bottom plate 7 are in smooth curved surface transition, so that the circular sealing covers 5 can be upwards extruded when the bottom plate 7 is inserted, and are tightly pressed.
After using this six orifice plates, can take bottom plate 7 out, then take out circular closing cap 5 below each cultivation hole 4, can wash the inside of inverted circular truncated cone shape recess 2 very conveniently, through the sour processing of bubble, after drying again and utilizing ultraviolet sterilization, can also used repeatedly in some basic experiments that require relatively not high after accomplishing six orifice plates equipment, can practice thrift certain cost.
The circular closing cap 5 with be provided with cockscomb structure groove 51 between the culture plate body 1, cockscomb structure groove 51 sets up respectively circular closing cap 5 with on the culture plate body 1, on the circular closing cap 5 cockscomb structure groove 51 sets up at circular closing cap 5 upper end surface edge, on the culture plate body 1 cockscomb structure groove 51 is provided with culture plate body 1 with circular closing cap 5 junction surface.
The embodiment also provides a cell immunofluorescence staining method, which comprises the following steps:
s1: cell climbing sheet
The microscopic coverslips were placed into the inverted frustum-shaped recess with tweezers one by one in the clean bench, after which 1.2X 10 were placed5Planting Hep G2 cells in the modified six-well plate containing the microscopic cover glass in the inverted truncated cone-shaped groove, and allowing the cells to grow on the microscopic cover glass for 24 hours; because the diameter of the microscopic cover glass is larger than the diameter of the bottom end surface of the inverted frustum-shaped groove and smaller than the diameter of the opening at the upper end of the inverted frustum-shaped groove, the microscopic cover glass can be conveniently placed in the inverted frustum-shaped groove, the semicircular conical groove for inserting the tweezers is formed in the side edge of the inverted frustum-shaped groove, when cell culture is carried out, the microscopic cover glass is placed in the inverted frustum-shaped groove, and the microscopic cover glass can be clamped up by inserting the tweezers into the semicircular conical groove. The bottom end of the culture hole is provided with the inverted frustum-shaped groove, so that the microscopic cover glass can be conveniently placed in the groove;
s2: toxin treatment
The Hep G2 cells in the improved six-hole plate are continuously cultured for 24 hours, after the cells grow to a logarithmic growth phase, the original culture medium is removed, the DMEM complete culture medium containing MC-LR with different concentrations is used for culturing the cells for 24 hours, and the DMEM complete culture medium with the same volume is added into a control group, so that the difference between a toxin treatment group and the control group can be compared; when preparing MC-LR, firstly, 3 centrifuge tubes with 5mL are taken, corresponding to three concentrations of 1 μ M, 0.1 μ M and 0.01 μ M respectively, 2450 μ L, 2250 μ L of DMEM complete culture medium is added into the three centrifuge tubes respectively, 50 μ L of pure MC-LR stock solution is added into the centrifuge tube of a 1 μ M treatment group containing 2450 μ L of DMEM complete culture medium, and the blowing and sucking are uniform; then 250 mul of the mixed solution is taken from the centrifuge tube of the 1 mul processing group and added into the centrifuge tube of the 0.1 mul processing group containing 2250 mul of DMEM complete medium, and the mixture is evenly blown and sucked; then 250 mul of mixed solution is taken from the centrifuge tube of the 0.1 mul processing group and added into the centrifuge tube of the 0.01 mul processing group, and the blowing and the suction are uniform; finally, 2250. mu.L of 1. mu.M, 2250. mu.L of 0.1. mu.M and 2500. mu.L of 0.01. mu.M MC-LR working solutions were prepared, 2mL of each MC-LR working solution with different concentrations was added to the corresponding wells, and toxin-treated for 24 hours;
s3: cell fixation
Removing a toxin-containing culture medium by suction, cleaning the microscopic cover glass with a PBS (phosphate buffer solution) solution for 2 times, adding 1mL of a 3% glutaraldehyde solution into each culture well, fixing cells for 10 minutes at room temperature, removing a fixing solution in the improved six-well plate by suction, cleaning the microscopic cover glass with the PBS solution for 3 times, wherein the cleaning time is 5 minutes each time, and washing away residual glutaraldehyde;
s4: penetrating film
Adding 1mL of 1% transmembrane solution into each culture well, and incubating for 10 minutes at room temperature; after membrane penetration is finished, 1% of membrane penetrating liquid is sucked and discarded, and the membrane is washed for 1 time by PBS solution;
s5: sealing of
Adding 1mL of protein-free rapid blocking solution (1X) to each culture well, and blocking for 10 minutes;
s6: primary antibody incubation
Recovering the protein-free rapid blocking solution (1X), and subjecting the rabbit-derived γ -H2AX primary antibody to a reaction in the presence of the protein-free rapid blocking solution (1X) according to a ratio of 1: diluting by a multiple of 1000, adding an antibody diluent into the inverted frustum-shaped groove, stretching into the inverted frustum-shaped groove along the semicircular conical groove by using a pair of tweezers to clamp the microscopic cover glass, enabling one surface containing cells to be in contact with the antibody diluent, reversely buckling the inverted frustum-shaped groove, incubating at 37 ℃ for 30 minutes, stretching into the inverted frustum-shaped groove along the semicircular conical groove by using the pair of tweezers to clamp the microscopic cover glass, enabling one surface containing cells to face upwards, sucking and discarding the primary antibody diluent in the inverted frustum-shaped groove by using a pipettor, and cleaning the microscopic cover glass 6 times by using 0.2% PBST solution, wherein the cleaning time is 5 minutes each time so as to remove the residual primary antibody;
s7: incubation with secondary antibody
Alexa488 fluorescently labeled goat anti-rabbit secondary antibody was added to protein-free rapid blocking solution (1 ×) according to 1: diluting by a multiple of 1000, adding an antibody diluent into the inverted frustum-shaped groove, stretching the antibody diluent into the inverted frustum-shaped groove along the semicircular conical groove by using a pair of tweezers to clamp the microscopic cover glass, enabling one surface containing cells to be in contact with the antibody diluent, reversely buckling the antibody diluent in the inverted frustum-shaped groove, incubating the antibody diluent in a dark place at 37 ℃ for 20 minutes, stretching the antibody diluent into the inverted frustum-shaped groove along the semicircular conical groove by using the pair of tweezers to clamp the microscopic cover glass, enabling one surface containing cells to face upwards, sucking and discarding the primary anti-diluent in the inverted frustum-shaped groove by using a pipettor, and cleaning the microscopic cover glass 6 times by using 0.2% PBST solution in the dark place, wherein the cleaning time is 5 minutes each time so as to remove residual secondary antibodies;
s8: nuclear counterstaining
Staining the nucleus with 1 mu g/mL Hoechst33342 in the dark for 2 minutes, and washing 5 times in the dark with PBS solution, wherein the washing time is 5 minutes each time;
s9: color development
Dropping an anti-fluorescence quenching blocking piece on a glass slide, then reversely buckling one surface of the antibody-marked microscopic cover glass, which is provided with cells, on the anti-fluorescence quenching blocking piece, sucking and removing the redundant anti-fluorescence quenching blocking piece around the microscopic cover glass, and then observing fluorescence under a fluorescence microscope and taking a picture.
In step S1, before cell climbing, the microscopic glass slide is placed in a culture dish with a diameter of 100mm, ethanol with a purity of 75% is poured into the culture dish, the microscopic glass slide is slowly shaken for 24 hours on a shaking table, then placed in ultraviolet rays for irradiation for 30 minutes, finally washed for 2 times by a basic culture medium and then placed in the culture dish with a diameter of 100mm containing the basic culture medium, the periphery of the culture dish is sealed by a sealing film, and the culture dish is placed in a refrigerator with a temperature of 4 ℃ for soaking for more than 6 hours and stored for later use.
In steps S6 and S7, the antibody dilution was added in an amount of 60 μ L during both primary antibody incubation and secondary antibody incubation.
As shown in fig. 8-10, fig. 8, 9 and 10 show immunofluorescence staining maps for marking γ -H2AX lesion points by using a conventional cell immunofluorescence staining method, wherein fig. 8 shows staining maps of Hoechst33342 dye on cell nuclei, which can show that the cell nuclei have unclear outlines and overlap, fig. 9 shows staining maps of γ -H2AX lesion points in cell nuclei by using rabbit-derived γ -H2AX primary antibody and Alexa488 fluorescence-labeled goat anti-rabbit secondary antibody, in which white bright spots are all cells containing γ -H2AX lesion points, and fig. 10 shows synthesis maps of fig. 8 and 9, which can better locate whether the γ -H2AX lesion points are located in the cell nuclei.
Fig. 11, 12 and 13 show immunofluorescence staining patterns of injury spots marked by the cellular immunofluorescence staining method provided in the first embodiment of the present invention, wherein fig. 11 is a staining pattern of Hoechst33342 dye on cell nuclei, which shows clear outline of cell nuclei and well-defined layers between cell nuclei, fig. 12 is a staining pattern of injury spots of γ -H2AX in cell nuclei using rabbit-derived γ -H2AX primary antibody and Alexa488 fluorescently labeled goat anti-rabbit secondary antibody, in which white bright spots are cells containing injury spots of γ -H2 AX. FIG. 13 is the composite map of FIGS. 11 and 12, which is constructed to better locate the location of the lesion site γ -H2AX in the nucleus.
Since the cellular immunofluorescence staining method and parameters thereof in the second embodiment and the first embodiment are not changed, the implementation result is the same as that in the first embodiment, please refer to the first embodiment, which is not repeated herein.
From the above experiments, according to the steps S5, S6, and S7 of the immunofluorescence staining method of the present invention, the speed of the experiment operation of blocking and antibody incubation can be increased, the generation of immunofluorescence map background can be reduced, the staining effect on cells is good, the number of cells can be accurately judged according to the counted number of cell nuclei, the position of the γ -H2AX damage point can be easily determined, and the damaged cells can be located.
In conclusion, the inverted circular truncated cone-shaped grooves are formed in the bottom ends of the culture holes of the six-hole plate in the two groups of embodiments, so that the microscopic cover slips can be conveniently placed in the six-hole plate, the bottom surfaces of the microscopic cover slips cannot be directly contacted with the bottom surfaces of the inverted circular truncated cone-shaped grooves, the contact force between the microscopic cover slips and the bottom surfaces of the culture holes is reduced, the trouble that the microscopic cover slips are difficult to take out when being directly attached to the bottom surfaces of the culture holes is avoided, and the situation that the microscopic cover slips are broken possibly caused by frequent taking out and putting back is also avoided; the reserved groove is formed in the side edge of the inverted truncated cone-shaped groove, so that the microscopic cover glass can be conveniently taken and placed through tweezers during an immunofluorescence experiment, the operation time of an experimenter is saved, and the working efficiency of the experimenter is improved; the immunofluorescence staining method in the two groups of embodiments is simple and convenient to operate and easy to master, and the used 3% glutaraldehyde fixing solution has a good fixing and penetrating effect on cells; the protein-free rapid sealing liquid has good sealing effect and high sealing speed, is particularly suitable for sealing phosphorylated antigens, saves time and reduces the background of a fluorescent picture; the antibody is diluted by the recovered protein-free rapid blocking solution (1X), so that the nonspecific binding of the antibody can be reduced, and the cost of using the protein-free rapid blocking solution (1X) is saved; compared with the PBST solution with the concentration of 0.1%, the PBST solution with the concentration of 0.2% has better cleaning effect on residual primary antibody and secondary antibody; compared with the membrane penetration solution with the concentration of 0.5%, the membrane penetration solution with the concentration of 1% has better effect of increasing the permeability of cell membranes.
Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. In the description of the present invention, "a plurality" means at least two, e.g., two, three, etc., unless specifically limited otherwise.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction. Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
Claims (10)
1. An improvement six orifice plates which characterized in that: including cultivateing plate body, lid and six cultivation holes, six the opening of cultivateing the hole all upwards, and its array distribution is in cultivate on the plate body, the lid sets up cultivate the upper end of plate body, detachably covers cultivate on the plate body, cultivate the bottom of hole and seted up the ascending inverted frustum of a circle recess of opening on the surface, the diameter of microscopic cover glass is greater than the bottom surface diameter of inverted frustum of a circle recess is less than the upper end opening diameter of inverted frustum of a circle recess, the reserve tank that is used for inserting tweezers is offered to the side reason of inverted frustum of a circle recess, and when carrying out cell culture, microscopic cover glass arranges in the inside of inverted frustum of a circle recess is through inserting tweezers can be pressed from both sides microscopic cover glass in the reserve tank and get and put.
2. The improved six-well plate of claim 1, wherein: six culture holes are distributed on the culture plate body according to two rows and three columns, and the distance between every two adjacent culture holes is equal.
3. The improved six-well plate of claim 1, wherein: the preformed groove can be a triangular conical groove, and the triangular conical groove extends downwards along the side edge of the inverted frustum-shaped groove until the bottom end surface of the inverted frustum-shaped groove is stopped.
4. The improved six-well plate of claim 1, wherein: the preformed groove can also be a semicircular conical groove, and the semicircular conical groove extends downwards along the side edge of the inverted frustum-shaped groove until the bottom end surface of the inverted frustum-shaped groove is cut off.
5. The improved six-well plate of claim 1, wherein: the improved six-hole plate further comprises a bottom plate and a circular sealing cover, wherein the circular sealing cover is arranged at the bottom end of each inverted truncated cone-shaped groove and seals the bottom end of each inverted truncated cone-shaped groove, the bottom plate is inserted into the lower end of the culture plate body and is positioned at the lower end of the circular sealing cover, and the circular sealing cover is upwards extruded.
6. The improved six-well plate of claim 5, wherein: the culture plate is characterized in that a serrated groove is arranged between the circular sealing cover and the culture plate body, the serrated groove is respectively arranged on the circular sealing cover and the culture plate body, the serrated groove on the circular sealing cover is arranged on the edge of the surface of the circular sealing cover, and the serrated groove on the culture plate body is arranged on the surface of the joint of the culture plate body and the circular sealing cover.
7. The cell immunofluorescence staining method based on the improved six-well plate according to any one of claims 1 to 6, comprising the following steps:
s1: cell climbing sheet
Respectively placing a plurality of microscopic cover slips into the inverted frustum-shaped grooves by using forceps, uniformly planting cells into the inverted frustum-shaped grooves in the improved six-hole plate containing the microscopic cover slips, and allowing the cells to grow on the microscopic cover slips in an adherent manner for 24 hours;
s2: toxin treatment
After the cells grow to a logarithmic growth phase, the original culture medium is sucked and discarded, the cells are cultured for 24 hours by using a DMEM complete culture medium containing MC-LR with different concentrations, and an isovolumetric DMEM complete culture medium is added into a control group;
s3: cell fixation
Removing a culture medium containing toxin in the improved six-hole plate by suction, cleaning the microscopic cover glass for 2 times by using a PBS (phosphate buffer solution), adding 1mL of a 3% glutaraldehyde solution into each culture hole, fixing cells for 10 minutes at room temperature, removing a fixing solution in the improved six-hole plate by suction, and cleaning the microscopic cover glass for 3 times by using the PBS solution, wherein the cleaning time is 5 minutes each time;
s4: penetrating film
Adding 1mL of 1% membrane penetrating liquid into each culture well, and incubating for 10 minutes at room temperature, wherein the membrane penetrating liquid is 1mL of Triton X-100 dissolved in 100mL of PBS solution; after membrane penetration is finished, 1% of membrane penetrating liquid is sucked and discarded, and the membrane is washed for 1 time by PBS solution;
s5: sealing of
Adding 1mL of protein-free rapid blocking solution into each culture well, and blocking for 10 minutes;
s6: primary antibody incubation
After recovery of the protein-free rapid blocking solution, rabbit-derived γ -H2AX primary antibody was treated with the protein-free rapid blocking solution according to a 1: diluting by a multiple of 1000, adding an antibody diluent into the inverted-truncated-cone-shaped groove, stretching into the inverted-truncated-cone-shaped groove along the reserved groove by using a pair of tweezers to clamp the microscopic cover slip, enabling one surface containing cells to be in contact with the antibody diluent, reversely buckling the groove in the inverted-truncated-cone-shaped groove, incubating for 30 minutes at 37 ℃, stretching into the inverted-truncated-cone-shaped groove along the reserved groove by using the pair of tweezers to clamp the microscopic cover slip, enabling one surface containing cells to face upwards, sucking and discarding the primary antibody diluent in the inverted-truncated-cone-shaped groove by using a pipette, cleaning the microscopic cover slip for 6 times by using 0.2% PBST solution, dissolving the 0.2% PBST solution which is 200 mu L Tween-20 in 100mL PBS, and cleaning for 5 minutes each time so as to remove residual primary antibody;
s7: incubation with secondary antibody
Alexa488 fluorescence-labeled goat anti-rabbit secondary antibody was added to the protein-free rapid blocking solution according to the ratio of 1: diluting by a multiple of 1000, adding an antibody diluent into the inverted-truncated-cone-shaped groove, stretching into the inverted-truncated-cone-shaped groove along the reserved groove by using a pair of tweezers to clamp the microscopic cover glass, enabling one surface containing cells to be in contact with the antibody diluent, reversely buckling the inverted-truncated-cone-shaped groove, incubating at 37 ℃ in a dark place for 20 minutes, stretching into the inverted-truncated-cone-shaped groove along the reserved groove by using the pair of tweezers to clamp the microscopic cover glass, enabling one surface containing cells to face upwards, sucking and discarding the secondary antibody diluent in the inverted-truncated-cone-shaped groove by using a pipettor, cleaning the microscopic cover glass 6 times in the dark place by using 0.2% PBST solution, wherein the cleaning time is 5 minutes each time so as to remove residual;
s8: nuclear counterstaining
Staining the nucleus with 1 mu g/mL Hoechst33342 in the dark for 2 minutes, and washing the nucleus with PBS solution in the dark for 5 times, wherein the washing time is 5 minutes each time;
s9: color development
Dropping an anti-fluorescence quenching blocking piece on a glass slide, then reversely buckling one side of the antibody-labeled microscopic cover glass, which is provided with cells, on the anti-fluorescence quenching blocking piece, sucking away the redundant anti-fluorescence quenching blocking piece around the microscopic cover glass, and then observing and photographing under a fluorescence microscope.
8. The cellular immunofluorescent staining method according to claim 7, wherein: in step S1, before cell climbing, the microscopic glass slide is placed in a culture dish with a diameter of 100mm, ethanol with a purity of 75% is poured into the culture dish, the culture dish is slowly shaken for 24 hours on a shaking table, then placed in ultraviolet rays for irradiation for 30 minutes, finally washed for 2 times by a basic culture medium, then placed in the culture dish with a diameter of 100mm containing the basic culture medium, sealed for the periphery by a sealing film, placed in a refrigerator with a temperature of 4 ℃ for soaking for more than 6 hours, and stored for later use.
9. The cellular immunofluorescent staining method according to claim 7, wherein in the step S2, the process of formulating MC-LR comprises the steps of:
s21: firstly, 3 centrifuge tubes with the volume of 5mL are taken, the centrifuge tubes respectively correspond to three concentrations of 1 MuM, 0.1 MuM and 0.01 MuM, 2450 MuM DMEM complete culture medium, 2250 MuL DMEM complete culture medium and 50 MuM pure MC-LR stock solution are respectively added into the centrifuge tubes of a 1 MuM treatment group containing 2450 MuL DMEM complete culture medium, and the aspiration is uniform;
s22: then 250 mul of the mixed solution is taken from the centrifuge tube of the 1 mul processing group and added into the centrifuge tube of the 0.1 mul processing group containing 2250 mul of DMEM complete medium, and the mixture is evenly blown and sucked; then 250 mu L of mixed solution is taken from the centrifuge tube of the 0.1 mu M processing group to the centrifuge tube of the 0.01 mu M processing group, and the mixture is evenly blown and sucked;
s23: finally, 2250. mu.L of 1. mu.M, 2250. mu.L of 0.1. mu.M and 2500. mu.L of 0.01. mu.M MC-LR working solutions were prepared, and 2mL of each MC-LR working solution with different concentrations was added to the corresponding wells and subjected to toxin treatment for 24 hours.
10. The cellular immunofluorescent staining method according to claim 7, wherein: in steps S6 and S7, the antibody dilution was added in an amount of 60 μ L during both primary antibody incubation and secondary antibody incubation.
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|---|---|---|---|---|
| CN111500454A (en) * | 2020-04-29 | 2020-08-07 | 盛小龙 | Cell slide, cell slide pickup device, cell culture plate and application method |
| CN114807022A (en) * | 2022-03-29 | 2022-07-29 | 上海市第一人民医院 | Method for establishing cavernous fibrosis disease model |
-
2019
- 2019-12-11 CN CN201911267151.2A patent/CN110923141A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111500454A (en) * | 2020-04-29 | 2020-08-07 | 盛小龙 | Cell slide, cell slide pickup device, cell culture plate and application method |
| CN111500454B (en) * | 2020-04-29 | 2023-07-21 | 湖南省肿瘤医院 | Cell climbing sheet, cell climbing sheet pickup device, cell culture plate and application method |
| CN114807022A (en) * | 2022-03-29 | 2022-07-29 | 上海市第一人民医院 | Method for establishing cavernous fibrosis disease model |
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