CN118330207A - Fixing agent for improving fluorescent background in fixed cell immunofluorescence method and method for fixing cells by using fixing agent - Google Patents
Fixing agent for improving fluorescent background in fixed cell immunofluorescence method and method for fixing cells by using fixing agent Download PDFInfo
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
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- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
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Abstract
The invention belongs to the technical field of biological and medical engineering, and discloses a fixing agent for improving fluorescent background in fixed cell immunofluorescence method and a method for fixing cells, wherein the fixing agent is prepared by dissolving paraformaldehyde in a glycyl-L-glutamic acid-containing solution, and the prepared fixing agent at least meets the following conditions: the concentration of the paraformaldehyde is 10-20 g/L; the concentration of glycyl-L-glutamic acid is 1-2 g/L; wherein the content of glycyl-L-glutamic acid is 1/10 of that of paraformaldehyde; the osmotic pressure is 800-2400 mOsm/L; the pH value is 7.0-7.6. The fixing agent can realize cell fixation under the action of high osmotic pressure, and simultaneously realize protection of cell membrane structure and antigen conformation of over-expressed protein inside and outside cell membrane, and can also prevent cell autolysis and lysis phenomenon of cells in the fixation process, and can solve the problems of high nonspecific fluorescence background and low fluorescence contrast of cell climbing sheets.
Description
Technical Field
The invention relates to a fixing agent for improving a fluorescence background in a fixed cell immunofluorescence method and a method for fixing cells by using the fixing agent, in particular to a fixing agent capable of reducing the whole nonspecific fluorescence background of a fixed cell slide in the cell immunofluorescence method and improving fluorescence contrast, and a method for fixing cells by using the fixing agent, belonging to the technical field of biological and medical engineering.
Background
Cell immunofluorescence (CBA) is a technique used for clinical antibody disease diagnosis, and is an indirect immunofluorescence method based on cell transfection, and the principle of action is that a human antigen gene is transfected into cells, so that the cells express antigen proteins in a large amount, living cells or cells are fixed on a slide to prepare detection materials, antibodies (primary antibodies) in a patient specimen are specifically combined with the antigens, then a secondary antibody with fluorescent markers is combined with the primary antibodies, and the slide interpretation result is read according to the specific fluorescent conditions observed under a fluorescent microscope, wherein the method for fixing the cells is the fixed cell immunofluorescence method (Fixed cell based assay, F-CBA).
In the preparation process of the conventional cell immunofluorescence method fixed climbing sheet, a fixing agent for fixing cells is usually prepared from paraformaldehyde or formaldehyde solution and phosphate buffer solution isotonic with cells, and the principle of fixing tissues and cells is that the paraformaldehyde can be chemically crosslinked with amino groups in the cells to form a reticular structure, so that proteins in the cells are fixed on a certain section within a certain time. However, when cells are fixed by using paraformaldehyde, the conformation of some sites of the cell surface proteins may be changed after fixation, so that the antibodies cannot bind to generate false negatives, or nonspecific binding (false positives) may be generated at other sites due to the fixation, which may cause nonspecific fluorescence background in the F-CBA cell slide.
The fixing solution (i.e. the buffered saline solution containing paraformaldehyde solution or formaldehyde) currently used for the cell climbing sheet has a plurality of defects, such as: the concentration of the existing paraformaldehyde solution or formaldehyde solution for fixing cells is 1-4%, and the common buffer salt solution for the paraformaldehyde solution is as follows: 1) Sodium dihydrogen phosphate 1.9mM, disodium hydrogen phosphate 8.1mM; 2) 0.1M phosphate buffer. When the fixative is used for fixing the cell climbing plate, nonspecific combination of paraformaldehyde inevitably generates nonspecific fluorescence background, so that the prepared cell climbing plate has reduced sensitivity when being used for immunofluorescence detection of low-concentration antibody samples, particularly serum samples with lower antibody concentration. In addition, cell samples immobilized with paraformaldehyde must be detected in a short period of time, for example, some CBA cells immobilized with paraformaldehyde require detection within 4 hours, otherwise adverse effects such as tandem dye degradation, which affect fluorescence intensity, may occur.
However, for the cell climbing tablet prepared in large quantities, if the cell climbing tablet needs to be used for a long time, the F-CBA cell climbing tablet needs to be stored in a refrigerator at 4 ℃ or-20 ℃ for a long time, and in the freezing process, the nonspecific fluorescent background in the climbing tablet fixed by paraformaldehyde can be further increased, and meanwhile, due to long-term storage or freezing treatment, the fluorescent contrast can be reduced along with the storage time, so that the sensitivity and accuracy in the film reading process are reduced.
Unlike hypotonic or isotonic (280-310 mOsm/L) fixatives, when an ultra-high osmotic pressure (800-2400 mOsm/L) is used as the fixative, the ultra-high osmotic pressure causes cell shrinkage and increases fluorescence intensity, but the contrast is lower instead than that of a hypotonic or isotonic fixative because the nonspecific fluorescent background and the specific fluorescent signal on the cell surface are increased simultaneously. Therefore, to improve the sensitivity and accuracy of immobilized cell immunofluorescence (F-CBA) immobilized slide detection, it is desirable to provide an immobilization formulation that reduces the background of nonspecific fluorescence and improves fluorescence contrast. In the prior art, the invention patent with the publication number of CN108169474A provides a novel cell fixing agent applied to immunofluorescence technology, and the L-ascorbic acid is adopted to soak a cell climbing tablet with water or PBS buffer solution or physiological saline solution, so that the antigen form of the cell climbing tablet can be well preserved, the specific combination with a corresponding antibody is facilitated, and meanwhile, when the L-ascorbic acid is used for fixing cells, the cell can be subjected to certain permeation treatment, so that no cell permeation agent is required to be additionally added when the cells are fixed, and the L-ascorbic acid is adopted to replace paraformaldehyde and the like, so that the cell climbing tablet is nontoxic and can avoid the generation of toxic and harmful substances. Although L-ascorbic acid has a series of advantages as described above, L-ascorbic acid still needs to be formulated with a solution that is isotonic or lower than the intracellular osmotic pressure when used in a cell fixative, and this patent does not describe whether the use of L-ascorbic acid can improve the non-specific fluorescence background of paraformaldehyde and the like when used in a cell slide when used in immunofluorescence detection, nor does it describe whether the fixative can improve fluorescence contrast, and thus ensure the sensitivity and accuracy of the detection results, and neither the patent nor the prior art gives corresponding data support.
Disclosure of Invention
The invention aims to provide a fixing agent for improving the fluorescent background in a fixed cell immunofluorescence method, which adopts a combination of paraformaldehyde and glycyl-L-glutamic acid to prepare the fixing agent with ultrahigh osmotic pressure, can enable cell membrane and intracellular proteins to be rapidly fixed, simultaneously protect cell membrane structures under high osmotic pressure and antigen conformations of over-expressed proteins inside and outside the cell membrane, can also prevent cells from autolysis and lysis in the fixing process, and can avoid the increase of the non-specific fluorescent background and the decrease of fluorescent contrast of F-CBA cell climbing sheets in subsequent immunofluorescence experiments.
The invention also aims to provide a method for fixing cells by using the fixing agent, and the cell slide obtained by the method can realize the effects of a lower F-CBA cell slide nonspecific fluorescence background and higher fluorescence contrast in immunofluorescence experiments even though the cell slide is subjected to cryopreservation.
The invention is realized by the following technical scheme: a fixative for reducing the fluorescent background of a fixed cell slide, which is prepared by dissolving paraformaldehyde in a glycyl-L-glutamic acid-containing solution, wherein the solution is at least one selected from water, a buffer solution or physiological saline, and the prepared fixative at least meets the following conditions:
1) Wherein the concentration of the paraformaldehyde is 10-20 g/L;
2) Wherein the concentration of glycyl-L-glutamic acid is 1-2 g/L;
3) Wherein the content of glycyl-L-glutamic acid is 1/10 of that of paraformaldehyde;
4) The osmotic pressure is 800-2400 mOsm/L;
5) The pH value is 7.0-7.6.
When the fixative is used for fixing membrane proteins, the osmotic pressure of the fixative is controlled to be 1920-2400 mOsm/L.
When the fixative is used for fixing intracellular proteins, the osmotic pressure of the fixative is controlled to be 800-960 mOsm/L.
The solution included NaCl, KCl, na 2HPO4 and KH 2PO4.
The concentration of NaCl, KCl, na 2HPO4 and KH 2PO4 in the fixing agent is 20-60 g/L, 0.5-1.5 g/L, 3.6-10.8 g/L and 0.6-1.8 g/L in sequence.
A method for fixing cells by using the fixing agent comprises the steps of soaking and fixing the cells for 20-30 minutes by using the fixing agent, cleaning the cells, performing post-treatment, airing the cells, and freezing the cells for later use.
When washing cells, the cells may be washed 3 times with PBS for 5 minutes each.
The post-treatment comprises sealing treatment, when the fixing agent is used for fixing membrane proteins, sealing liquid is added into the membrane proteins after cleaning, sealing treatment is carried out for 30-60 minutes, the sealing liquid is absorbed, and after the cell climbing slices are dried, the cell climbing slices are frozen at the temperature of minus 20 ℃ for later use.
The post-treatment further comprises membrane permeation treatment, when the fixative is used for fixing intracellular proteins, after washing, cell lysate is added into the intracellular proteins for membrane permeation treatment for 5 minutes, then sealing liquid is added, sealing treatment is carried out for 30-60 minutes, sealing liquid is absorbed, and after the cell climbing slices are dried, the cell climbing slices are frozen at the temperature of minus 20 ℃ for later use.
The blocking solution is a bovine serum albumin solution, such as PBS+5% BSA.
The cell lysate is Triton X-100, such as Triton X-100 at 0.2% concentration.
Compared with the prior art, the invention has the following advantages:
(1) The invention creatively adopts the formulation of the ultra-high osmotic compound paraformaldehyde fixing agent, utilizes the ultra-high osmotic pressure solution with the conventional osmotic pressure of 3-8 times to prepare the paraformaldehyde solution, and simultaneously adds glycyl-L-glutamic acid as a protective agent and a specific fluorescent signal enhancer, thereby realizing cell fixation under the action of high osmotic pressure, simultaneously realizing the protection of cell membrane structures and the antigen conformation of over-expressed proteins inside and outside the cell membrane, and also preventing the phenomena of autolysis and lysis of cells in the fixing process.
(2) The invention can solve the problems of the increase of the nonspecific fluorescence background and the decrease of the fluorescence contrast of the F-CBA cell climbing sheet when the existing formaldehyde or paraformaldehyde fixative formula is used for fixing cells, adopts paraformaldehyde and glycyl-L-glutamic acid as specific combination for the first time, is dissolved in a solution with ultrahigh osmotic pressure in a specific concentration range, and can solve the problems of the increase of the nonspecific fluorescence background and the decrease of the fluorescence contrast of the F-CBA cell climbing sheet, thereby improving the sensitivity and the accuracy of detection.
(3) The fixative is suitable for fixation of membrane proteins and intracellular proteins, can be operated by adopting different osmotic pressures and paraformaldehyde/glycyl-L-glutamic acid concentrations according to different cell structures, and is simple to operate.
(4) The method can realize the preparation of a large number of cell climbing sheets, the cell climbing sheets treated by the method can be stored in a freezing environment for standby, and experiments prove that the cell climbing sheets can still provide lower background and higher sensitivity in immunofluorescence experiments than the common fixing method even though the cell climbing sheets are stored in a freezing way.
Drawings
FIG. 1 is an immunofluorescence image (before freezing) of the membrane proteins immobilized on CHO cells in example 1, example 2, comparative examples 1 to 4.
FIG. 2 is an immunofluorescence image (after freezing) of the membrane proteins immobilized on CHO cells in example 1, example 2, comparative examples 1 to 4.
FIG. 3 is an immunofluorescence image (before freezing) of the case of example 3, example 4, comparative example 5 to comparative example 8 in which intracellular proteins were immobilized on CHO cells.
FIG. 4 is an immunofluorescence image (before freezing) of the case of example 3, example 4, comparative example 5 to comparative example 8 in which intracellular proteins were immobilized on CHO cells.
FIG. 5 is an immunofluorescence image of the immobilization of membrane proteins on HEK293 cells in example 5.
FIG. 6 is an immunofluorescence image of the case of immobilization of intracellular proteins on HEK293 cells as described in example 6.
Detailed Description
The present invention will be described in further detail with reference to examples, but embodiments of the present invention are not limited thereto.
Fixative for membrane proteins, cell fixation method, immunofluorescence experiment and contrast experiment:
Example 1: ultra-high permeability compound paraformaldehyde fixing agent
The ultra-high permeability compound paraformaldehyde fixing agent is prepared according to the following formula dosage (see table 1), namely the fixing agent for improving the fluorescent background of the cell slide in the fixed cell immunofluorescence method.
When in preparation, a proper amount of paraformaldehyde is dissolved in NaCl, KCl, na 2HPO4、KH2PO4 and glycyl-L-glutamic acid aqueous solution to obtain the product. The osmotic pressure of the prepared fixing agent is 2400 mOsm/L, and the pH value is 7.0-7.6.
TABLE 1
Example 2: ultra-high permeability compound paraformaldehyde fixing agent
An ultra-high permeability compound paraformaldehyde fixing agent was prepared in the same manner as in example 1. The formulation amounts are shown in Table 2. The osmotic pressure of the prepared fixative is 2400mOsm/L, and the pH value is 7.0-7.6.
TABLE 2
Comparative example 1: isotonic fixative
Conventional isotonic paraformaldehyde fixatives were prepared and the formulation amounts are shown in table 3. The osmotic pressure of the prepared fixative is 320mOsm/L, and the pH value is 7.0-7.6.
TABLE 3 Table 3
Comparative example 2: hypertonic fixative
The formulation dosage of the prepared hypertonic paraformaldehyde fixing agent is shown in Table 4. The osmotic pressure of the prepared fixative is 2400mOsm/L, and the pH value is 7.0-7.6.
TABLE 4 Table 4
Comparative example 3: hypertonic fixative
The formulation dosage of the prepared hypertonic paraformaldehyde fixing agent is shown in Table 5. The osmotic pressure of the prepared fixative is 2400mOsm/L, and the pH value is 7.0-7.6.
TABLE 5
Comparative example 4: ultra-high-permeability compound paraformaldehyde fixing agent added with spermidine
The amount of spermidine added to the formulation of example 1 is shown in Table 6. The osmotic pressure of the prepared fixative is 2400mOsm/L, and the pH value is 7.0-7.6.
TABLE 6
The fixatives of the above examples 1, 2, comparative examples 1 to 4 were subjected to membrane protein fixation.
Taking an anti-aquaporin 4 (Aquaporin 4) antibody (hereinafter referred to as AQP 4) as an example, the anti-AQP 4 antibody is one of autoimmune antibodies, and the AQP4 is a membrane protein, and is related to diseases such as neuromyelitis optica and demyelination.
Overexpression of the AQP4 gene on CHO cells, stable cells were selected for the following:
CHO cells stably expressing AQP4 and wild-type CHO cells were mixed, inoculated into 96-well plates, grown to complete confluence, and then cell-fixed.
The prepared fixative is soaked and fixed on cells for 20 minutes, the cells are washed by PBS for 3 times for 5 minutes each time, then PBS plus 5 percent BSA is added for sealing treatment for 45 minutes, sealing liquid is absorbed and removed, and the cell slide is frozen at the temperature of minus 20 ℃ for standby.
Immunofluorescence experiments were performed on the F-CBA cell slide prepared in example 1, example 2, comparative example 1 to comparative example 4, respectively, including before and after freezing (30 days of freezing).
The immunofluorescence experimental method is as follows:
preparing a blood or cerebrospinal fluid sample, wherein the sample diluent comprises the following components:
phosphate buffer (pH 7.0-7.4), BSA (2%);
Carrying out primary measurement dilution on a sample by using a sample diluent, wherein the dilution ratio of serum is 1:10, and stock solution (undiluted) is adopted for cerebrospinal fluid;
sample incubation: incubation at 37 ℃ for 1 hour, washing 3 times with PBS;
Dilution of the secondary antibodies with sample dilutions: adopting goat anti-human IgG (H+L), wherein the dilution ratio is 1:500, incubating for 45 minutes at room temperature, and washing for 3 times by PBS;
and (3) interpretation: negative and positive samples were read under a microscope.
The experimental results are shown in fig. 1 and 2.
In FIG. 1, the fluorescence patterns expressed by the cells of example 1, example 2, comparative example 1 to comparative example 4, respectively, from left to right, are shown as a secondary anti-fluorescence pattern, and the two fluorescence patterns are shown as co-located overlapping images.
As can be seen from FIG. 1, the immobilization agents of example 1 and example 2 have the best immobilization effect on membrane proteins, and the fluorescent contrast is high, and no specific fluorescent background appears; the fluorescent effect of example 1 and example 2 are the same, and it can be confirmed that the fixative of the present invention has a stable effect when used for fixation of membrane proteins, and has good consistency in parallel experiments. From its secondary anti-fluorescence intensity and background, the fluorescence effects of example 1, example 2, comparative example 4 were identical and optimal, and the fluorescence effects of comparative examples 1 to 3 were significantly worse than those of example 1, example 2, and comparative example 4. Comparative example 4 shows the same fluorescence effects as in examples 1 and 2, and shows that the addition of spermidine as a cell membrane protecting agent to the fixative of the present invention has no effect.
In FIG. 2, the fluorescence patterns expressed by the cells of example 1, example 2, comparative example 1 to comparative example 4, respectively, from left to right, are shown as a secondary anti-fluorescence pattern, and the two fluorescence patterns are shown as co-located overlapping images.
As can be seen from fig. 2, the fixing effect of the fixing agent of the present invention on the membrane protein is not affected after the cell slide is frozen.
Fixative for intracellular proteins, cell fixation method, immunofluorescence experiment and contrast experiment:
example 3: ultra-high permeability compound paraformaldehyde fixing agent
An ultra-high permeability compound paraformaldehyde fixative was prepared in the same manner as in example 1, with the formulation amounts shown in Table 7. The osmotic pressure of the prepared fixative is 960mOsm/L, and the pH value is 7.0-7.6.
TABLE 7
Example 4: ultra-high permeability compound paraformaldehyde fixing agent
An ultra-high permeability compound paraformaldehyde fixative was prepared in the same manner as in example 1, with the formulation amounts shown in Table 8. The osmotic pressure of the prepared fixative is 960mOsm/L, and the pH value is 7.0-7.6.
TABLE 8
Comparative example 5: isotonic fixative
The conventional isotonic paraformaldehyde fixative is prepared, and the formulation dosage is shown in Table 9. Osmotic pressure of the fixing agent after preparation is
320MOsm/L, and the pH value is 7.0-7.6.
TABLE 9
Comparative example 6: hypertonic fixative
The formulation dosage of the prepared hypertonic paraformaldehyde fixing agent is shown in Table 10. The osmotic pressure of the prepared fixative is 960mOsm/L, and the pH value is 7.0-7.6.
Table 10
Comparative example 7: hypertonic fixative
The formulation dosage of the prepared hypertonic paraformaldehyde fixing agent is shown in Table 11. Osmotic pressure of the fixing agent after preparation is
960MOsm/L, pH value is 7.0-7.6.
TABLE 11
Comparative example 8: ultra-high-permeability compound paraformaldehyde fixing agent added with spermidine
Spermidine was added to the formulation of example 5 in the amounts shown in Table 12. The osmotic pressure of the prepared fixative is 960mOsm/L, and the pH value is 7.0-7.6.
Table 12
The fixatives of examples 3, 4, comparative examples 5 to 8 above were used for intracellular protein fixation.
Taking an anti-glutamate decarboxylase 65 (glutamic acid decarboxylase) antibody (hereinafter abbreviated as GAD 65) as an example, the anti-GAD 65 antibody is one of autoimmune antibodies, and GAD65 is an intracellular protein, and is associated with diseases such as stiff human syndrome, cerebellar ataxia, chronic epilepsy, limbic encephalitis, and the like.
Overexpression of the GAD65 gene on CHO cells, stable cells were selected for the following operations:
CHO cells stably expressing GAD65 and wild-type CHO cells were mixed, inoculated into 96-well plates, grown to complete confluence, and then fixed.
The prepared fixative is soaked and fixed on cells for 30 minutes, the cells are washed by PBS for 3 times for 5 minutes, triton X-100 with 0.2% concentration is added for membrane permeation treatment for 5 minutes, the cells are washed by PBS for 3 times for 5 minutes, PBS plus 5% BSA is added for 5 minutes, the sealing treatment is carried out for 45 minutes, sealing liquid is absorbed, and the cells are frozen in-20 ℃ for standby after the cell climbing slices are dried.
Immunofluorescence experiments were performed on the F-CBA cell slide prepared in example 3, example 4, comparative example 5 to comparative example 8, respectively, including before and after freezing (30 days of freezing).
The immunofluorescence experimental method is as follows:
preparing a blood or cerebrospinal fluid sample, wherein the sample diluent comprises the following components:
phosphate buffer (pH 7.0-7.4), BSA (2%);
Carrying out primary measurement dilution on a sample by using a sample diluent, wherein the dilution ratio of serum is 1:10, and stock solution (undiluted) is adopted for cerebrospinal fluid;
sample incubation: incubation at 37 ℃ for 1 hour, washing 3 times with PBS;
Dilution of the secondary antibodies with sample dilutions: adopting goat anti-human IgG (H+L), wherein the dilution ratio is 1:500, incubating for 45 minutes at room temperature, and washing for 3 times by PBS;
and (3) interpretation: negative and positive samples were read under a microscope.
The experimental results are shown in fig. 3 and 4.
In FIG. 3, the fluorescence patterns expressed by the cells of examples 3, 4,5 and 8, respectively, from left to right, are shown as a secondary anti-fluorescence pattern and a co-located image of the two patterns.
As can be seen from FIG. 3, the immobilization agents of example 3 and example 4 have the best effect on intracellular proteins, and the fluorescent contrast is high, and no specific fluorescent background appears; the fluorescence effects of example 3 and example 4 are the same, and it can be confirmed that the fixative of the present invention has a stable effect when used for intracellular protein fixation and has good consistency in parallel experiments. From its secondary anti-fluorescence intensity and background, the fluorescence effects of example 3, example 4, comparative example 8 were identical and optimal, and the fluorescence effects of comparative examples 5 to 7 were significantly worse than those of example 3, example 4, and comparative example 8. Comparative example 8 shows the same fluorescence effects as in examples 3 and 4, and shows that the addition of spermidine as a cell membrane protecting agent to the fixative of the present invention has no effect.
In FIG. 4, the fluorescence patterns expressed by the cells of example 3, example 4, comparative example 5 to comparative example 8, respectively, from left to right, are shown as a secondary anti-fluorescence pattern, and the two fluorescence patterns are shown as co-located overlapping images.
The fixative of the present invention is not limited to CHO cell lines but is also applicable to other cell lines.
Example 5: hypertonic paraformaldehyde compound fixative, membrane protein fixation and cellular immunofluorescence (HEK 293 cells)
In this example, an anti-AQP 4 antibody was used as an example for the fixation of membrane proteins and cellular immunofluorescence by using a hypertonic paraformaldehyde compound fixative for HEK293 cells.
An ultra-high permeability compound paraformaldehyde fixative was prepared in the same manner as in example 1, with the formulation amounts shown in Table 13. The osmotic pressure of the prepared fixing agent is 2400 mOsm/L, and the pH value is 7.0-7.6.
TABLE 13
Overexpression of the AQP4 gene on HEK293 cells, stable transgenic cells were selected for the following:
HEK293 cells stably expressing AQP4 and wild HEK293 cells were mixed, inoculated into 96-well plates, grown to complete confluence, and then fixed.
The prepared fixative is soaked and fixed on cells for 30 minutes, the cells are washed by PBS for 3 times, each time is 5 minutes, PBS and 5% BSA are blocked for 45 minutes, blocking liquid is sucked and removed, and immunofluorescence operation is carried out after the slide is dried.
Immunofluorescence:
preparing a blood or cerebrospinal fluid sample, wherein the sample diluent comprises the following components:
phosphate buffer (pH 7.0-7.4), BSA (2%);
carrying out primary measurement dilution on a sample by using a sample diluent, wherein the dilution ratio is as follows: serum: 1:10, cerebrospinal fluid: a stock solution;
sample incubation: incubation at 37 ℃ for 1 hour, washing 3 times with PBS;
dilution of the secondary antibodies with sample dilutions: sheep anti-human IgG (H+L), 1:500, incubated for 45 min at room temperature, washed 3 times with PBS;
and (3) interpretation: negative and positive samples were read under a microscope.
The experimental results are shown in FIG. 5.
In FIG. 5, the left to right images correspond to the fluorescence image, the secondary anti-fluorescence image, and the co-located images of the two images expressed by the cells. As can be seen from FIG. 5, when the fixative of the present invention is used to fix membrane proteins on HEK293 cells, the fluorescence contrast is high and no specific fluorescent background appears.
Example 6: hypertonic paraformaldehyde compound fixative, intracellular protein fixation and cellular immunofluorescence (HEK 293 cells)
Taking an anti-GAD 65 antibody as an example, an embodiment of fixing intracellular proteins and cellular immunofluorescence by using a hypertonic paraformaldehyde compound fixative for HEK293 cells is performed.
An ultra-high permeability compound paraformaldehyde fixative was prepared in the same manner as in example 1 with the formulation amounts shown in Table 14. The osmotic pressure of the prepared fixing agent is 960 mOsm/L, and the pH value is 7.0-7.6.
TABLE 14
Overexpression of the GAD65 gene on HEK293 cells, stable transgenic cells were selected for the following:
293 cells stably expressing GAD65 and wild-type 293 cells were mixed, inoculated into 96-well plates, grown to complete confluence, and then fixed.
The prepared fixative is soaked and fixed on cells for 30 minutes, the cells are washed by PBS for 3 times for 5 minutes, the cells are treated by adding 0.4% Triton X-100 to pass through a membrane for 5 minutes, the cells are washed by PBS for 3 times for 5 minutes, PBS and 5% BSA are blocked for 45 minutes, the blocking liquid is sucked and removed, and immunofluorescence operation is carried out after the slide is dried.
Immunofluorescence:
preparing a blood or cerebrospinal fluid sample, wherein the sample diluent comprises the following components:
phosphate buffer (pH 7.0-7.4), BSA (2%);
Carrying out primary measurement dilution on a sample by using a sample diluent, wherein the dilution ratio of serum is 1:10, and stock solution (undiluted) is adopted for cerebrospinal fluid;
sample incubation: incubation at 37 ℃ for 1 hour, washing 3 times with PBS;
Dilution of the secondary antibodies with sample dilutions: adopting goat anti-human IgG (H+L), wherein the dilution ratio is 1:500, incubating for 45 minutes at room temperature, and washing for 3 times by PBS;
and (3) interpretation: negative and positive samples were read under a microscope.
The experimental results are shown in FIG. 6.
In FIG. 6, the left to right images correspond to the fluorescence image, the secondary anti-fluorescence image, and the co-located images of the two images expressed by the cells. As can be seen from FIG. 6, when the fixative of the present invention is used to fix intracellular proteins on HEK293 cells, the fluorescence contrast is high and no specific fluorescent background appears.
The foregoing description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in any way, and any simple modification, equivalent variation, etc. of the above embodiment according to the technical matter of the present invention fall within the scope of the present invention.
Claims (10)
1. A fixative for improving the fluorescent background in a fixed cell immunofluorescence method, characterized in that: the fixing agent is prepared by dissolving paraformaldehyde in a glycyl-L-glutamic acid-containing solution, wherein the solution is at least one selected from water, a buffer solution and physiological saline, and the prepared fixing agent at least meets the following conditions:
1) Wherein the concentration of the paraformaldehyde is 10-20 g/L;
2) Wherein the concentration of glycyl-L-glutamic acid is 1-2 g/L;
3) Wherein the content of glycyl-L-glutamic acid is 1/10 of that of paraformaldehyde;
4) Osmotic pressure is 800-2400 mOsm/L;
5) The pH value is 7.0-7.6.
2. A fixative as set forth in claim 1 wherein: when the fixative is used for fixing membrane proteins, the osmotic pressure of the fixative is controlled to be 1920-2400 mOsm/L.
3. A fixative as set forth in claim 1 wherein: when the fixative is used for fixing intracellular proteins, the osmotic pressure of the fixative is controlled to be 800-960 mOsm/L.
4. A fixative as set forth in claim 1 wherein: the solution included NaCl, KCl, na 2HPO4 and KH 2PO4.
5. The fixative of claim 4, wherein: the concentration of NaCl, KCl, na 2HPO4 and KH 2PO4 in the fixing agent is 20-60 g/L, 0.5-1.5 g/L, 3.6-10.8 g/L and 0.6-1.8 g/L in sequence.
6. A method of fixing cells using the fixative of any one of claims 1-5, wherein: and (3) soaking and fixing the cells for 20-30 minutes by using a fixing agent, and cleaning, post-treating and airing the cells, and then freezing the cells for later use.
7. The method according to claim 6, wherein: the post-treatment comprises sealing treatment, when the fixing agent is used for fixing membrane proteins, sealing liquid is added into the membrane proteins after cleaning, sealing treatment is carried out for 30-60 minutes, the sealing liquid is absorbed, and after the cell climbing slices are dried, the cell climbing slices are frozen at the temperature of minus 20 ℃ for later use.
8. The method according to claim 7, wherein: the post-treatment further comprises membrane permeation treatment, when the fixative is used for fixing intracellular proteins, after washing, cell lysate is added into the intracellular proteins for membrane permeation treatment for 5 minutes, then sealing liquid is added, sealing treatment is carried out for 30-60 minutes, sealing liquid is absorbed, and after the cell climbing slices are dried, the cell climbing slices are frozen at the temperature of minus 20 ℃ for later use.
9. The method according to claim 7, wherein: the blocking solution is bovine serum albumin solution.
10. The method according to claim 8, wherein: the cell lysate is TritonX-100.
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