Antibody diluent for cell immunofluorescent staining

Abstract

The invention belongs to the technical field of biology, and particularly relates to an antibody diluent for cell immunofluorescent staining. In the antibody diluent, the volume fraction of glycerol is 5-20%, the mass fraction of bovine serum albumin is 2-5%, the mass fraction of sodium thiomersalate is 0.01-0.05%, and the molar concentration of a phosphate buffer solution is 0.01 M. The composition proportion of bovine serum albumin realizes a good sealing effect; triton X-100 is not used, so that an abnormal dyeing result caused by excessive permeation of cells is prevented; glycerin and sodium thiomersalate are added, and the preservation condition of 4 DEG C is matched, so that the antibody decomposition prevention effect of most existing products is achieved, the bacterial pollution prevention effect of long-term preservation can be achieved, the secondary antibody can be diluted without being prepared and used once in each experiment, and the secondary antibody can be prepared and used for more than six months at a time. Aiming at the antibody diluent, a special staining scheme is designed for a cell immunofluorescence staining method, and a good, stable and low-background staining effect can be realized.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and specifically relates to an antibody diluent for cell immunofluorescence staining. Background Technology

[0002] Currently, most of the diluents available on the market for diluting antibodies are secondary antibody diluents, with a small amount of primary antibody diluents. The main components are bovine serum albumin (BSA) combined with protein stabilizers. Their main purpose is to reduce non-specific binding staining and delay antibody protein degradation.

[0003] Existing antibody diluents on the market are developed with the aim of achieving both effective blocking to reduce non-specific binding and staining, and extending the shelf life of diluted secondary antibodies to eliminate the need for pre-preparation for each experiment, thus improving convenience. To achieve this blocking effect, almost all such products use bovine serum albumin (BSA) as the blocking agent. The principle is that BSA occupies non-specific binding sites on the surface of carriers (such as cell culture plates or cell slides), reducing the adsorption or binding of non-target molecules during experiments, thereby improving the specificity and accuracy of the experiment. BSA is chosen because it can interact with a variety of material surfaces, making it widely applicable, and its chemical properties are relatively stable, making it less susceptible to changes in blocking performance due to experimental conditions. Furthermore, BSA's structure is rich in hydrophobic groups, allowing it to interact with hydrophobic surfaces for blocking, and its moderate isoelectric point minimizes charge interference, resulting in minimal interference with subsequent detection results.

[0004] In addition, to extend the shelf life of diluted secondary antibodies, many products incorporate substances such as Triton X-100 and protein stabilizers. The specific copolymers added vary, but the common goal is to delay antibody degradation. Furthermore, some existing products also include detergents or anticoagulants in their formulation.

[0005] The purpose of detergents is to reduce interfering proteins in the sample and should also help regulate pH. Anticoagulants are used because some reagents target whole blood samples, and the incubation process requires anticoagulation. However, a common problem with both is that they are not designed for immunofluorescence staining of cells, and their addition can damage cell membranes.

[0006] Existing antibody diluents have the following problems: 1. Most existing antibody diluents include protein stabilizers to delay antibody degradation. However, in actual use, it has been found that the main reason why secondary antibodies diluted with 0.01M phosphate buffer can only be used for a short time (mostly no more than one day) is not protein degradation, but rather that bacterial contamination is more likely to occur after dilution of secondary antibodies for more than one day.

[0007] 2. Most existing antibody diluents used for immunofluorescence staining are not designed specifically for cell immunofluorescence methods. Most are designed for ELISA staining, blot staining, dot staining, etc., and most of them contain Triton X-100, which has cell membrane permeability and can significantly interfere with staining results. Summary of the Invention

[0008] This invention provides an antibody diluent for cell immunofluorescence staining, which solves the above-mentioned problems.

[0009] The technical solution of this invention is implemented as follows: An antibody diluent for cellular immunofluorescence staining, specifically designed for cellular immunofluorescence staining, comprising glycerol, bovine serum albumin, sodium thimerosal, and phosphate buffer.

[0010] Through the above technical solution, the antibody diluent of this invention contains only these four components and does not contain any components that interfere with cell staining. This solves the problem that existing antibody diluents cannot be used for cell immunofluorescence staining, as they can damage cell membranes and lead to severe non-specific staining. The antibody diluent of this invention considers both blocking and preservation effects, is specifically designed for cell staining, and can even be used on live cells without causing damage.

[0011] Optionally, the volume fraction of glycerol is 5-20%, the mass fraction of bovine serum albumin is 2-5%, the mass fraction of sodium thimerosal is 0.01-0.05%, and the molar concentration of phosphate buffer is 0.01M.

[0012] Using the above technical solutions, if the volume fraction of glycerol is too low, the cryoprotection effect during low-temperature storage will be insufficient; if it is too high, the fluidity of the dilution will decrease. If the mass fraction of bovine serum albumin is too low, the blocking effect will be poor; if it is too high, the staining results will have high background fluorescence. If the mass fraction of sodium thimerosal is too low, the preservative effect will be insufficient; if it is too high, it will damage cells.

[0013] Optionally, the volume fraction of glycerol is 9.09%.

[0014] Optional, the mass fraction of bovine serum albumin is 3%.

[0015] Optionally, the mass fraction of sodium thimerosal is 0.03%.

[0016] A method for preparing an antibody diluent for cell immunofluorescence staining includes the following steps: S1. Dispense 0.01M phosphate buffer into clean conical flasks and sterilize at 120°C for 30 minutes; S2. After weighing bovine serum albumin and sodium thimerosal, add them to a sterilized conical flask containing 0.01M phosphate buffer and shake on a shaker at room temperature for 30 minutes at 60 rpm. S3. Dispense glycerol into the above-mentioned well-mixed reagent using a dispenser, and shake again on a shaker at room temperature at 60 r / min for 10 minutes. S4. Dilute the prepared antibody diluent with secondary antibody on day 1 and day 10 after preparation. Then perform immunofluorescence staining on the diluted secondary antibody on day 1 and day 10 after dilution.

[0017] A method for using an antibody diluent for cell immunofluorescence staining includes the following steps: A1. Warming: Remove the overexpressing antigen cell plate, 0.01M phosphate buffer, secondary antibody, and antibody dilution solution from the 4°C freezer and warm them to room temperature for 10-15 minutes before staining. A2. Primary antibody dilution: Dilute the sample to be tested 1:10 using antibody diluent as a solvent; A3. Sample primary antibody incubation: Add 1:10 sample primary antibody to each well and incubate at 37°C for 1 hour; A4. Washing: Remove the primary antibody from the 1:10 sample in each well, add 0.01M phosphate buffer to each well, and wash on a shaker at medium speed for 5 minutes. Repeat the washing process 3 times. A5. Secondary antibody dilution: Dilute the secondary antibody 1:500 using antibody dilution buffer as a solvent; A6. Secondary antibody incubation: Add 1:500 secondary antibody to each well and incubate at 37°C for 30 min; A7. Washing: Remove the 1:500 secondary antibody from the wells, add 0.01M phosphate buffer to each well, and wash on a shaker at medium speed for 5 minutes. Repeat the washing process 3 times. A8. Add 0.01M phosphate buffer to each well and observe the experimental results under a fluorescence microscope.

[0018] By using the above technical solution, the antibody diluent of the present invention is used in accordance with the above-described method for cell immunofluorescence staining. On the one hand, the diluted secondary antibody can be stored for a long time without needing to be re-diluted each time. On the other hand, the cell wells or slides stained using this method can be stored in the dark for more than a week and can be reviewed repeatedly at any time. Furthermore, it contains blocking components, eliminating the need for a separate blocking operation step and saving experimental time.

[0019] Optionally, steps A6-A8 require light protection.

[0020] Through the above technical solution, the light-shielding treatment in steps A6-A8 avoids the decomposition of the fluorescent secondary antibody upon exposure to light.

[0021] After adopting the above technical solution, the beneficial effects of the present invention are: The antibody diluent of this invention is specifically designed for use in immunofluorescence staining of cells, and a specific staining scheme is designed for its application. This antibody diluent eliminates most of the interfering components found in existing technologies, does not damage cell membranes, does not cause non-specific staining, and achieves good blocking effects while being easy to store. This antibody diluent is specifically designed for immunofluorescence staining of cells and can even be used for staining live cells without causing cell damage.

[0022] The antibody diluent of this invention, with its bovine serum albumin composition ratio, achieves excellent blocking effect; the antibody diluent does not use reagents such as Triton X-100, preventing abnormal staining results caused by excessive cell permeability; the addition of glycerol and sodium thimerosal, combined with storage conditions at 4°C, not only achieves the anti-antibody decomposition effect of most existing products, but also achieves anti-bacterial contamination effect for long-term storage, so that the secondary antibody dilution does not need to be prepared for each experiment, but can be prepared once and used for more than six months.

[0023] This invention provides a specialized staining protocol for antibody dilution solutions, designed specifically for cell immunofluorescence staining, which achieves good, stable, and low-background staining results. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 The effect of staining cell membranes to express antibodies in Example 1; Figure 2 The effect of staining cytoplasm to express antibodies in Example 1; Figure 3 Example 2 shows the effect of staining cell membranes to express antibodies; Figure 4 This is to demonstrate the effect of staining cytoplasm to express antibodies in Example 2; Figure 5 Example 3 shows the effect of staining cell membranes to express antibodies; Figure 6 Example 3 shows the effect of staining cytoplasm to express antibodies; Figure 7 To show the effect of antibody expression on the cell membrane during staining in Comparative Example 2; Figure 8 To show the effect of antibody expression in the cytoplasm of stained cells in Comparative Example 2; Figure 9To show the effect of antibody expression on the cell membrane in Comparative Example 3; Figure 10 To show the effect of antibody expression in the cytoplasm of stained cells in Comparative Example 3. Detailed Implementation

[0026] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] This invention discloses an antibody diluent for cell immunofluorescence staining, which includes glycerol, bovine serum albumin, sodium thimerosal, and phosphate buffer; wherein the volume fraction of glycerol is 5-20%, the mass fraction of bovine serum albumin is 2-5%, the mass fraction of sodium thimerosal is 0.01-0.05%, and the molar concentration of phosphate buffer is 0.01M.

[0028] The functions of glycerol are: (1) to lower the freezing point of diluted secondary antibodies and prevent ice crystal formation caused by small temperature fluctuations under 4℃ storage conditions; (2) to reduce microbial metabolism and prevent bacterial contamination in conjunction with 4℃ storage conditions and sodium thimerosal.

[0029] The functions of bovine serum albumin are: (1) to achieve the blocking effect on the detection carrier and reduce non-specific binding staining; (2) to delay the decomposition of antibody protein when stored at 4℃.

[0030] The function of sodium thimerosal: as a preservative, it works in conjunction with storage conditions of 4°C and glycerin to reduce microbial metabolism and prevent bacterial contamination.

[0031] The function of 0.01M phosphate buffer: to adapt to cell immunofluorescence staining, provide a stable and appropriate pH and osmotic pressure, and prevent abnormal damage to the cell structure used for detection from affecting the staining results.

[0032] The antibody diluent of this invention is used to dilute the antibody according to the required dilution ratio and then store it in the dark at 4°C. The shelf life can reach more than six months. If high-frequency use is required, it is recommended to aliquot the antibody and minimize the time exposed to light when opening the storage container each time it is used.

[0033] Example 1 An antibody diluent for immunofluorescence staining of cells, comprising glycerol, bovine serum albumin, sodium thimerosal, and phosphate buffer; wherein the volume fraction of glycerol is 5%, the mass fraction of bovine serum albumin is 2%, the mass fraction of sodium thimerosal is 0.01%, and the molar concentration of phosphate buffer is 0.01M.

[0034] A method for preparing an antibody diluent for cell immunofluorescence staining includes the following steps: S1. Dispense 450mL of 0.01M phosphate buffer into a 500mL clean conical flask, and sterilize using a vertical autoclave set to 120℃ for 30 minutes. S2. In a clean area, weigh 15.0g of bovine serum albumin and 0.15g of sodium thimerosal using an electronic balance, add them to a sterilized conical flask containing 450mL of 0.01M phosphate buffer, and shake on a shaker at room temperature at 60r / min for 30 minutes to mix thoroughly. S3. In a clean area, dispense 50 mL of glycerin into the already mixed reagent using a dispenser, and shake again on a shaker at room temperature at 60 r / min for 10 minutes to mix thoroughly. S4. Finished Product Quality Control: The prepared antibody diluent was diluted with secondary antibody on day 1 and day 10 after preparation. The diluted secondary antibody was then subjected to immunofluorescence staining on day 1 and day 10 after dilution. The results should be bright with low background and the staining results should be stable after several stainings.

[0035] Example 2 An antibody diluent for immunofluorescence staining of cells comprises glycerol, bovine serum albumin, sodium thimerosal, and phosphate buffer; wherein the volume fraction of glycerol is 9.09%, the mass fraction of bovine serum albumin is 3%, the mass fraction of sodium thimerosal is 0.03%, and the molar concentration of phosphate buffer is 0.01M.

[0036] A method for preparing an antibody diluent for cell immunofluorescence staining includes the following steps: S1. Dispense 450mL of 0.01M phosphate buffer into a 500mL clean conical flask, and sterilize using a vertical autoclave set to 120℃ for 30 minutes. S2. In a clean area, weigh 15.0g of bovine serum albumin and 0.15g of sodium thimerosal using an electronic balance, add them to a sterilized conical flask containing 450mL of 0.01M phosphate buffer, and shake on a shaker at room temperature at 60r / min for 30 minutes to mix thoroughly. S3. In a clean area, dispense 50 mL of glycerin into the already mixed reagent using a dispenser, and shake again on a shaker at room temperature at 60 r / min for 10 minutes to mix thoroughly. S4. Finished Product Quality Control: The prepared antibody diluent was diluted with secondary antibody on day 1 and day 10 after preparation. The diluted secondary antibody was then subjected to immunofluorescence staining on day 1 and day 10 after dilution. The results should be bright with low background and the staining results should be stable after several stainings.

[0037] Example 3 An antibody diluent for cellular immunofluorescence staining comprises glycerol, bovine serum albumin, sodium thimerosal, and phosphate buffer; wherein the volume fraction of glycerol is 20%, the mass fraction of bovine serum albumin is 5%, the mass fraction of sodium thimerosal is 0.05%, and the molar concentration of phosphate buffer is 0.01M.

[0038] A method for preparing an antibody diluent for cell immunofluorescence staining includes the following steps: S1. Dispense 450mL of 0.01M phosphate buffer into a 500mL clean conical flask, and sterilize using a vertical autoclave set to 120℃ for 30 minutes. S2. In a clean area, weigh 15.0g of bovine serum albumin and 0.15g of sodium thimerosal using an electronic balance, add them to a sterilized conical flask containing 450mL of 0.01M phosphate buffer, and shake on a shaker at room temperature at 60r / min for 30 minutes to mix thoroughly. S3. In a clean area, dispense 50 mL of glycerin into the already mixed reagent using a dispenser, and shake again on a shaker at room temperature at 60 r / min for 10 minutes to mix thoroughly. S4. Finished Product Quality Control: The prepared antibody diluent was diluted with secondary antibody on day 1 and day 10 after preparation. The diluted secondary antibody was then subjected to immunofluorescence staining on day 1 and day 10 after dilution. The results should be bright with low background and the staining results should be stable after several stainings.

[0039] Example 4 The method of using the antibody diluent for cell immunofluorescence staining in this invention is as follows, with a general-purpose 96-well cell culture plate as an example. The staining process is basically the same when changing to different cell carriers, only the appropriate amount of each liquid needs to be changed.

[0040] A1. Warming: The overexpressing antigen cell well plates and related reagents (0.01M phosphate buffer, secondary antibody, antibody dilution solution) were taken out of the 4℃ refrigerator and warmed to room temperature for 10-15 minutes before staining. A2. Primary antibody dilution: Dilute the sample to be tested 1:10 using antibody diluent as a solvent; A3. Primary antibody incubation: Add 60 μL of the corresponding 1:10 primary antibody to each well and incubate at 37°C for 1 h. A4. Washing: Remove the primary antibody from the 1:10 sample in each well, add 150 μL of 0.01 M phosphate buffer to each well, and wash on a shaker at medium speed for 5 min. Wash a total of 3 times. A5. Secondary antibody dilution: Dilute the secondary antibody 1:500 using antibody dilution buffer (using Alexa Fluor-labeled goat anti-human IgG secondary antibody as an example). A6. Secondary antibody incubation: Add 60 μL of 1:500 secondary antibody to each well and incubate at 37°C for 30 min; Note: This step requires protection from light.

[0041] A7. Washing: Remove the 1:500 secondary antibody from the wells, add 150 μL of 0.01 M phosphate buffer to each well, and wash on a shaker at medium speed for 5 min. Repeat the washing process 3 times. A8. Add 150 μL of 0.01 M phosphate buffer to each well and observe the experimental results under a fluorescence microscope.

[0042] Comparative Example 1 An antibody diluent for immunofluorescence staining of cells, comprising glycerol, bovine serum albumin, sodium thimerosal, and phosphate buffer; wherein the volume fraction of glycerol is 3%, the mass fraction of bovine serum albumin is 1%, the mass fraction of sodium thimerosal is 0.005%, and the molar concentration of phosphate buffer is 0.005M.

[0043] Comparative Example 2 An antibody diluent for cellular immunofluorescence staining comprises glycerol, bovine serum albumin, sodium thimerosal, and phosphate buffer; wherein the volume fraction of glycerol is 25%, the mass fraction of bovine serum albumin is 8%, the mass fraction of sodium thimerosal is 0.07%, and the molar concentration of phosphate buffer is 0.02M.

[0044] Comparative Example 3 An antibody diluent for cell immunofluorescence staining, comprising glycerol, bovine serum albumin, sodium thimerosal, and phosphate buffer; wherein the volume fraction of glycerol is 5%, the mass fraction of bovine serum albumin is 2%, the mass fraction of sodium thimerosal is 0.01%, the molar concentration of phosphate buffer is 0.01M, and the volume fraction of Triton X-100 is 0.1%.

[0045] The verification experiment is shown below: I. Verification of differences between Examples 1, 2, 3, and 4 and comparative examples The diluent formulations in Examples 1-3 are all within the technical concentration range of this scheme, and the effects are as expected. The usage method is the same as in Example 4.

[0046] The control group was treated according to Comparative Example 1. The storage time of the secondary antibody was significantly shortened after dilution, but it was still better than the above 0.01M PBS solution dilution scheme. The control group was treated according to Comparative Example 2. The resulting reagent could stain positive, but the contrast was poor and the cell morphology was obviously damaged. The control group was treated according to Comparative Example 3. The staining of plasma expression antibody was almost unaffected, but the membrane expression antibody could not be stained and the cells were obviously broken.

[0047] II. Staining Methods 1. Select 3 antibody projects (MOG, AQP4, GFAP), and dilute each project with commercially purified antibody to 3 titers (1:32, 1:100, 1:320). Pre-amplify the samples into 20 μL tubes, and use only one tube for each staining to avoid repeated freeze-thaw cycles that may cause a decrease in titer. 2. Dilute the secondary antibody 1:500 with the reagents of Examples 1-3 and Comparative Examples 1-3. Perform cell immunofluorescence staining every 4 days according to Example 4. Observe the staining results with a fluorescence inverted microscope. Fix the exposure time at 350ms and the exposure gain at 100%. The time of failure is defined as when no antibody or titer can be clearly seen under these conditions. The experiment is double-parallel.

[0048] Table 1. Statistics on the number of days of failure of secondary antibodies after dilution in Examples 1-3 and Comparative Examples 1-3. The average number of days of staining failure for MOG, AQP4, and GFAP in Example 1 were 186, 200, and 206, respectively; the average number of days of staining failure for MOG, AQP4, and GFAP in Example 2 were 192, 210, and 212, respectively; and the average number of days of staining failure for MOG, AQP4, and GFAP in Example 3 were 208, 244, and 232, respectively. All average values ​​were higher than 180 days, and the shortest single failure time was also greater than 180 days, achieving the expected results.

[0049] In Comparative Example 1, the average number of days of staining failure for MOG, AQP4, and GFAP were 48, 62, and 66, respectively, all below 180 days. In Comparative Example 2, the average number of days of staining failure for MOG, AQP4, and GFAP were 164, 178, and 186, respectively, with some items below 180 days, and cell morphology damage was visible during staining. In Comparative Example 3, staining with antibodies expressing the cell membrane resulted in severe cell membrane damage, making staining impossible, but the antibody expression in the stained plasma was basically normal.

[0050] analyze Figure 1-6 The staining was performed according to Examples 1-3. The staining results showed that the membrane expression antibody stained the cell membrane with bright red fluorescence and the staining background was clean. The cytoplasm expression antibody stained the cell with filamentous fluorescence in some edge positions and the staining background was also clean. Both morphologies were standard expression morphologies.

[0051] contrast Figure 7 , 8 Following the comparison in Example 2, it was found that the staining contrast was poor and the cell morphology was obviously damaged. The cell edges of the membrane expression antibody stained parts were blurred, and the cell edges of the plasma expression antibody stained parts showed a membrane-like expression morphology.

[0052] contrast Figure 9 , 10Following the comparison example 3, the staining of plasma expression antibody was almost unaffected, while the membrane expression antibody could not be stained, resulting in obvious cell disruption and staining that showed homogeneous staining of the disrupted cells as a whole.

[0053] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the technical solution of the present invention should be included within the protection scope of the present invention.

Claims

1. An antibody diluent for cell immunofluorescence staining, characterized in that, This antibody diluent is specifically designed for cell immunofluorescence staining and includes glycerol, bovine serum albumin, sodium thimerosal, and phosphate buffer.

2. The antibody diluent for cell immunofluorescence staining according to claim 1, characterized in that, The volume fraction of glycerol is 5-20%, the mass fraction of bovine serum albumin is 2-5%, the mass fraction of sodium thimerosal is 0.01-0.05%, and the molar concentration of phosphate buffer is 0.01M.

3. The antibody diluent for cell immunofluorescence staining according to claim 1, characterized in that, The volume fraction of glycerol is 9.09%.

4. The antibody diluent for cell immunofluorescence staining according to claim 1, characterized in that, The mass fraction of bovine serum albumin is 3%.

5. The antibody diluent for cell immunofluorescence staining according to claim 1, characterized in that, The mass fraction of sodium thimerosal is 0.03%.

6. A method for preparing an antibody diluent for cell immunofluorescence staining, characterized in that, The method for preparing the antibody diluent as described in claim 1 includes the following steps: S1. Dispense 0.01M phosphate buffer into clean conical flasks and sterilize at 120°C for 30 minutes; S2. After weighing bovine serum albumin and sodium thimerosal, add them to a sterilized conical flask containing 0.01M phosphate buffer and shake on a shaker at room temperature for 30 minutes at 60 rpm. S3. Dispense glycerol into the above-mentioned well-mixed reagent using a dispenser, and shake again on a shaker at room temperature at 60 r / min for 10 minutes. S4. Dilute the prepared antibody diluent with secondary antibody on day 1 and day 10 after preparation. Then perform immunofluorescence staining on the diluted secondary antibody on day 1 and day 10 after dilution.

7. A method for using an antibody diluent for cell immunofluorescence staining, characterized in that, The method of using the antibody diluent as described in claim 1 includes the following steps: A1. Warming: Remove the overexpressing antigen cell plate, 0.01M phosphate buffer, secondary antibody, and antibody dilution solution from the 4°C freezer and warm them to room temperature for 10-15 minutes before staining. A2. Primary antibody dilution: Dilute the sample to be tested 1:10 using antibody diluent as a solvent; A3. Sample primary antibody incubation: Add 1:10 sample primary antibody to each well and incubate at 37°C for 1 hour; A4. Washing: Remove the primary antibody from the 1:10 sample in each well, add 0.01M phosphate buffer to each well, and wash on a shaker at medium speed for 5 minutes. Repeat the washing process 3 times. A5. Secondary antibody dilution: Dilute the secondary antibody 1:500 using antibody dilution buffer as a solvent; A6. Secondary antibody incubation: Add 1:500 secondary antibody to each well and incubate at 37°C for 30 min; A7. Washing: Remove the 1:500 secondary antibody from the wells, add 0.01M phosphate buffer to each well, and wash on a shaker at medium speed for 5 minutes. Repeat the washing process 3 times. A8. Add 0.01M phosphate buffer to each well and observe the experimental results under a fluorescence microscope.

8. A method of using the antibody diluent for cell immunofluorescence staining according to claim 7, characterized in that, Steps A6-A8 require protection from light.

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