CN114594245A - Cleaning buffer solution adapted to full-automatic immunohistochemical dyeing machine and preparation method thereof - Google Patents
Cleaning buffer solution adapted to full-automatic immunohistochemical dyeing machine and preparation method thereof Download PDFInfo
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- 238000004140 cleaning Methods 0.000 title claims abstract description 72
- 238000002360 preparation method Methods 0.000 title claims abstract description 21
- 239000007853 buffer solution Substances 0.000 title claims description 42
- 238000004043 dyeing Methods 0.000 title abstract description 14
- 230000002055 immunohistochemical effect Effects 0.000 title description 11
- 239000000243 solution Substances 0.000 claims abstract description 122
- 239000011534 wash buffer Substances 0.000 claims abstract description 103
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 58
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 56
- 239000008213 purified water Substances 0.000 claims abstract description 47
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims abstract description 45
- 239000011780 sodium chloride Substances 0.000 claims abstract description 28
- 238000011532 immunohistochemical staining Methods 0.000 claims abstract description 24
- 229920001213 Polysorbate 20 Polymers 0.000 claims abstract description 12
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims abstract description 12
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims abstract description 12
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 21
- 238000000034 method Methods 0.000 claims description 16
- 238000002156 mixing Methods 0.000 claims description 7
- 238000005303 weighing Methods 0.000 claims description 7
- 230000006978 adaptation Effects 0.000 claims description 2
- 230000000694 effects Effects 0.000 abstract description 15
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 abstract description 14
- 238000012360 testing method Methods 0.000 abstract description 4
- 239000007983 Tris buffer Substances 0.000 description 27
- 210000001519 tissue Anatomy 0.000 description 22
- 238000005406 washing Methods 0.000 description 10
- 239000003153 chemical reaction reagent Substances 0.000 description 9
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 8
- 230000008569 process Effects 0.000 description 8
- 210000004027 cell Anatomy 0.000 description 7
- 238000001514 detection method Methods 0.000 description 7
- 238000005057 refrigeration Methods 0.000 description 7
- 238000010186 staining Methods 0.000 description 7
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- 239000012895 dilution Substances 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
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- 102000004190 Enzymes Human genes 0.000 description 3
- 239000002585 base Substances 0.000 description 3
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- 238000009472 formulation Methods 0.000 description 3
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- 244000005700 microbiome Species 0.000 description 3
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- 230000003204 osmotic effect Effects 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- 239000012224 working solution Substances 0.000 description 3
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 2
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 2
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 2
- 230000000844 anti-bacterial effect Effects 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
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- 239000003513 alkali Substances 0.000 description 1
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Classifications
-
- G—PHYSICS
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
Abstract
A washing buffer solution adapted by a full-automatic immunohistochemical staining machine and a preparation method thereof are characterized in that: the washing buffer solution is formed by dissolving a prepared washing buffer solution A in purified water, wherein the washing buffer solution A comprises the following main components: tris (hydroxymethyl) aminomethane, sodium chloride, tween-20 and ProClin950, and the pH value range of the washing buffer solution A is pH 7.3-7.5. The technical scheme of this application has the use that can avoid sodium azide to guarantee uniformity, stability and the accuracy of testing result, can also improve the advantage of dyeing effect and cleaning performance.
Description
Technical Field
The application relates to the technical field of full-automatic immunohistochemical dyeing machines, in particular to a cleaning buffer solution adapted to a full-automatic immunohistochemical dyeing machine and a preparation method thereof.
Technical Field
The immunohistochemical technology is rapidly developed in recent years, and along with the application and popularization of a full-automatic immunohistochemical staining instrument, the requirement on the staining quality is increasingly improved. The sample cleaning plays an important role in the whole immunohistochemical staining process, and the quality of the cleaning effect can directly determine the quality of the final staining result. Because of the structural characteristics of the full-automatic staining instrument, the volume of a container for containing a cleaning buffer solution is small, the container cannot be used for completely soaking tissue slices in the cleaning buffer solution like a manual sample cleaning operation, and the full-automatic immunohistochemical staining instrument needs to be used under a small usage amount when the sample is cleaned, so that the effective cleaning area of a cleaning solution is further reduced, the cleaning effect is relatively poor, and the subsequent immunohistochemical staining effect can be influenced. Therefore, how to normally complete the sample cleaning process on the full-automatic immunohistochemical staining machine is a technical problem that must be solved by the full-automatic immunohistochemical staining machine.
The imported registration formula of some washing buffer solutions imported into the full-automatic immunohistochemical staining machine is as follows: tris buffered saline, surfactant, 0.1% sodium azide, etc., for sample washing, as well as providing a buffered environment to the overall system. However, since sodium azide is a well-known hazardous chemical reagent, there are many inconveniences in storage, transportation, and use, and it is very important to find an alternative reagent formulation.
If the technical problem of normally completing the sample washing process on the full-automatic immunohistochemical staining machine is to be well solved, the improvement of the washing buffer can be realized. Placing the improved washing buffer solution on a full-automatic immunohistochemical dyeing machine, wherein on one hand, the improved washing buffer solution needs to completely immerse the tissue slices and complete better washing under the condition of less using amount, namely the improved washing buffer solution has better uniform wettability; on the other hand, the improved washing buffer solution has the problems of stable use, namely the improved washing buffer solution has better stability of pH and better antibacterial preservation function no matter in refrigeration or room temperature, the function of the improved washing buffer solution cannot be influenced, the pH value of the improved washing buffer solution is kept stable, and the improved washing buffer solution can be stably preserved and used; finally, the formulation of the improved washing buffer, in particular avoiding the use of sodium azide, is prepared.
Therefore, further research can be used for preparing a formula of a cleaning buffer solution of a full-automatic immunohistochemical dyeing machine, particularly, the use of sodium azide is avoided, the consistency, stability and accuracy of detection results are guaranteed, the more excellent dyeing effect and cleaning effect are improved, and the technical problem to be solved urgently is still achieved.
Disclosure of Invention
This application is not enough to prior art's the aforesaid to provide one kind and can avoid the use of sodium azide to guarantee uniformity, stability and the accuracy of testing result, can also improve the washing buffer solution of dyeing effect and washing effect's full-automatic immunohistochemical dyeing machine adaptation.
In order to solve the technical problem, the technical scheme adopted by the application is as follows: a washing buffer solution adapted for a full-automatic immunohistochemical staining machine is formed by dissolving a prepared washing buffer solution A in purified water, and the main components of the washing buffer solution A comprise: tris (hydroxymethyl) aminomethane (Tris), sodium chloride, Tween-20 and ProClin950, and the pH value of the washing buffer solution A is in the range of pH 7.3-7.5.
Further, in the washing buffer adapted to the fully automatic immunohistochemical staining machine of the present application, the volume usage ratio of the washing buffer a to the purified water in the washing buffer is 1:9 (for example, 900mL of purified water is prepared corresponding to 100mL of washing buffer a); the pH value range of the washing buffer solution is 7.3-7.5.
Further, the washing buffer a described above in the present application comprises, per 1000 mL: 11.0-12.5 g of Tris (hydroxymethyl) aminomethane (Tris), 75.0-90.0 g of sodium chloride, 203.5-6.0 mL of Tween-203, 9502.5-5.0 mL of ProClin and the balance of purified water, wherein the pH value ranges from pH7.3 to 7.5.
Further, the washing buffer a described above in the present application comprises, per 1000 mL: 11.5-12.5 g of Tris (hydroxymethyl) aminomethane (Tris), 80.0-90.0 g of sodium chloride, 204.5-6.0 mL of Tween-3, 9503.0-5.0 mL of ProClin and the balance of purified water, wherein the pH value ranges from pH7.3 to 7.5.
Further, the washing buffer a described above in the present application comprises, per 1000 mL: 12.0-12.5 g of Tris (hydroxymethyl) aminomethane (Tris), 82.0-90.0 g of sodium chloride, 205.0-6.0 mL of Tween-205, 9503.5-5.0 mL of ProClin and the balance of purified water, wherein the pH value ranges from pH7.3 to 7.5.
Further, the washing buffer a described above in the present application comprises, per 1000 mL: 12.2-12.5 g of Tris (hydroxymethyl) aminomethane (Tris), 85.0-90.0 g of sodium chloride, 205.5-6.0 mL of Tween-205, 9504.0-5.0 mL of ProClin and the balance of purified water, wherein the pH value ranges from pH7.3 to 7.5.
The application also provides a preparation method of the washing buffer solution A, which comprises the following specific preparation steps:
(1) weighing Tris (hydroxymethyl) aminomethane (Tris) according to a ratio, dissolving with purified water, adding sodium chloride according to a formula amount, adding tween-20 after complete dissolution, and adding ProClin950 after complete dissolution;
(2) fixing the volume of the solution obtained in the step (1) to a rated capacity by using purified water to obtain a cleaning buffer solution;
(3) and (3) adjusting the pH value of the washing buffer solution in the step (2) to enable the pH value to be in the range of pH 7.3-7.5, and thus obtaining the washing buffer solution A.
Preferably, step (3) of adjusting the pH uses a 1M NaOH solution.
Preferably, the pH adjustment in step (3) is carried out using a 1M HCl solution.
The application also provides a preparation method of the cleaning buffer solution, which specifically comprises the following steps: and (3) measuring the obtained washing buffer solution A and purified water according to the volume ratio of 1:9 to obtain corresponding volumes, then adding the washing buffer solution A into pure water, uniformly mixing, and measuring the pH value within the range of pH 7.3-7.5.
The application has the advantages that:
1. ProClin950, which is a high-efficiency biological preservative, is introduced into a cleaning buffer solution of a full-automatic immunohistochemical staining instrument for the first time, and the addition of the ProClin950 can effectively control the growth of microorganisms in an in vitro diagnostic reagent, so that the cleaning buffer solution can be stably stored and used for a long time no matter under a refrigeration condition or a room temperature condition; in addition, ProClin950 did not affect the rate of binding of key enzymes to substrates in diagnostic reagents, such as: horseradish peroxidase (HRP), Alkaline Phosphatase (AP), and also does not affect the binding of the antibody; moreover, more importantly, the addition of ProClin950 replaces the use of sodium azide in the traditional cleaning solution matched with the full-automatic immunohistochemical staining machine, so that the safety of the cleaning buffer solution is greatly improved.
2. In the washing buffer solution of the application, an aqueous solution of Tris (hydroxymethyl) aminomethane (Tris) is used as the buffer solution; tris is a weak base with a pKa of 8.1 at room temperature (25 ℃); according to the buffering theory, the effective buffering range of the Tris buffer solution is between pH7.0 and 9.2, the pH of the aqueous solution of Tris alkali is about 10.5, hydrochloric acid is generally added to adjust the pH value to a required value, and the buffer solution with the pH value can be obtained; in addition, sodium chloride is added to serve as a pH regulator and is in mutual synergistic effect with Tris, and can effectively participate in acid-base balance regulation of a solution, so that the pH value of a washing buffer solution is more stable, and the osmotic pressure of tissue extracellular fluid can be effectively maintained, so that a tissue sample is prevented from being damaged.
3. In the washing buffer solution, Tween-20 in a proper proportion is mainly added to serve as a mild surfactant, so that the washing buffer solution has good affinity (good wettability) with tissue slices, has the effect of renaturation of antigens, can improve the recognition capability of specificity and reduce the nonspecific binding of antibodies, and can serve as an emulsifier to uniformly disperse other substances in the solution.
4. In the washing buffer solution, Tris, sodium chloride, Tween-20, ProClin950 and other substances are used as raw materials for preparing the washing buffer solution, the components are cooperated with each other, so that the obtained washing buffer solution has better wettability, the pH value of the washing buffer solution has better stability and better antibacterial preservation function, the washing buffer solution can carry out an antigen restoration process on tissue slices under less using amount, the washing buffer solution required by each tissue slice only needs about 150 microliters on average, the washing effect is good, and the washing buffer solution can be matched with a full-automatic immunohistochemical dyeing machine for use.
5. The required performances of the cleaning solution and the repairing solution are inconsistent, the repairing solution is matched with heating to change the protein structure of the tissue cells, and the pH environment of the cleaning solution is the most suitable pH environment range of the tissue cells, can not be heated, and can not change or damage the protein structure of the tissue cells; therefore, the pH value of the cleaning solution is very important, the pH value range of the cleaning buffer solution A is limited to be 7.3-7.5, the reaction system can be effectively maintained to be stable, the cleaning effect is achieved, and the structure of tissue protein molecules cannot be damaged; the pH range of the specific cleaning solution allows the tissue cells to perform functional reaction in a suitable reaction system.
6. The cleaning solution is used on a full-automatic immunohistochemical dyeing machine, reagent formulas required by different detection methodologies have specific requirements, and the requirements on the cleaning solution are different because a detection principle, a detection system, a detection sample, a detection process and other factors are different; according to the washing method, the washing liquid is washed on the immunohistochemical dyeing machine, the pH system is firstly ensured to be stable, the molecular structure of the tissue cell protein cannot be damaged, the wettability and the washing effect are ensured to be good, and the washing effect can be ensured under the condition of less using amount.
7. The cleaning buffer solution is formed by firstly preparing the cleaning buffer solution A and then dissolving the cleaning buffer solution A in purified water, and is not in the form of a cleaning solution which is directly prepared for final use in the traditional method, because the cleaning solution is relatively high in consumption in the actual instrument operation process, if the cleaning solution is directly provided in the form of 1 multiplied by cleaning solution, the purchase number of later-stage users is increased, more refrigerating space is required to be provided, and the packaging cost is also increased; the cleaning solution exists in a form of taking a cleaning buffer solution A as a concentrated solution, and is mixed and diluted with pure water in the using process; therefore, only the concentrated solution needs to be purchased in the actual purchasing process of a user, 1 bottle of the concentrated solution can be used as 10 bottles, the concentrated solution is very convenient to prepare, and the finally-used cleaning solution can be obtained without pH detection after the concentrated solution is directly mixed with pure water; therefore, the washing buffer solution a can be provided as a concentrated solution in this manner of the present application, and then diluted with pure water to be used as a final washing solution product.
8. The application also limits the dilution ratio of the concentrated solution (cleaning buffer solution A) to pure water to be 1:9, and the pH value after dilution is still between 7.3 and 7.5, so that the packaging cost of the product is saved; in addition, the ratio of 1:9 is a concentration ratio which is creatively obtained according to the solubility of each reagent in water in the formula and is relatively consistent with the performance of the product, if the concentration ratio is increased, the product is easy to separate out crystals under the refrigeration condition, the product is not a uniform solution, and the pH stability is influenced; therefore, the dilution ratio not only can realize the functions of saving refrigeration space and reducing cost by purchasing quantity, but also can keep the stability of pH, simplify the dilution step and prevent the use effect from being influenced by the crystallization of the concentrated solution.
9. The sodium chloride is added into the formula of the cleaning solution, and the component can be used as a pH regulator to participate in acid-base balance adjustment of the solution, so that the pH of the buffer solution is more stable, the osmotic pressure of the extracellular fluid of the tissue is maintained, and the tissue sample is prevented from being damaged; if sodium chloride is not added in the formula, the osmotic pressure of the extracellular fluid of the tissue cells cannot be maintained in the process of cleaning the sample, the structure of the tissue cells can be damaged, the pH stability of the diluted working solution can be influenced, and the use effect of the cleaning solution can be seriously influenced if the pH is not within the range of 7.3-7.5 probabilistically; the problem is effectively solved and the pH stability of the whole cleaning solution is maintained by adding the sodium chloride.
Detailed Description
The technical solutions in the embodiments of the present application will be described clearly and completely below, and it is obvious that the described embodiments are only preferred embodiments, not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.
Example 1
A washing buffer A: every 1000mL of wash buffer a included: 11.0g of Tris (hydroxymethyl) aminomethane (Tris), 75.0g of sodium chloride, 203.5mL of tween-203, 9502.5mL of ProClin and the balance of purified water, wherein the pH value range is 7.3-7.5.
A wash buffer: every 1000mL of 1 × Wash buffer contained: 100mL of washing buffer solution A and 900mL of purified water, wherein the pH value range of the washing buffer solution is 7.3-7.5.
The preparation method of the washing buffer solution A comprises the following steps: weighing Tris according to a ratio, dissolving Tris with purified water, adding sodium chloride according to a formula amount, adding Tween-20 after completely dissolving, adding ProClin950 after dissolving, finally fixing the volume to a rated capacity with purified water, and adjusting the pH value of a cleaning buffer solution by using a 1M NaOH solution or an HCl solution to ensure that the pH value is within a range of pH 7.3-7.5.
The preparation method of the washing buffer solution comprises the following steps: and (3) measuring the volumes of the cleaning buffer solution A and the purified water according to the proportion of 1:9, adding the cleaning buffer solution A in the formula amount into pure water, uniformly mixing, and measuring the pH value, wherein the pH value is in the range of pH 7.3-7.5.
Example 2
A washing buffer A: every 1000mL of wash buffer a included: 12.0g of Tris (hydroxymethyl) aminomethane (Tris), 82.0g of sodium chloride, 204.5mL of tween-204, 9504.0mL of ProClin and the balance of purified water, wherein the pH value ranges from pH7.3 to 7.5.
A wash buffer: every 1000mL of 1 × Wash buffer contained: 100mL of washing buffer solution A and 900mL of purified water, wherein the pH value range is 7.3-7.5.
The preparation method of the washing buffer solution A comprises the following steps: weighing Tris according to a ratio, dissolving Tris with purified water, adding sodium chloride according to a formula amount, adding Tween-20 after completely dissolving, adding ProClin950 after dissolving, finally fixing the volume to a rated capacity with purified water, and adjusting the pH value of a cleaning buffer solution by using a 1M NaOH solution or an HCl solution to ensure that the pH value is within a range of pH 7.3-7.5.
The preparation method of the cleaning buffer solution comprises the following steps: and (3) measuring the volumes of the cleaning buffer solution A and the purified water according to the proportion of 1:9, adding the cleaning buffer solution A in the formula amount into pure water, uniformly mixing, and measuring the pH value, wherein the pH value is in the range of pH 7.3-7.5.
Example 3
A washing buffer A: every 1000mL of wash buffer a included: 12.5g of Tris (hydroxymethyl) aminomethane (Tris), 90.0g of sodium chloride, 206.0mL of Tween-2, 9505.0mL of ProClin and the balance of purified water, wherein the pH value ranges from pH7.3 to 7.5.
A wash buffer: every 1000mL of 1 × Wash buffer included: 100mL of washing buffer solution A and 900mL of purified water, wherein the pH value range is 7.3-7.5.
The preparation method of the washing buffer solution A comprises the following steps: weighing Tris according to a ratio, dissolving Tris with purified water, adding sodium chloride according to a formula amount, adding Tween-20 after completely dissolving, adding ProClin950 after dissolving, finally fixing the volume to a rated capacity with purified water, and adjusting the pH value of a cleaning buffer solution by using a 1M NaOH solution or an HCl solution to ensure that the pH value is within a range of pH 7.3-7.5.
The preparation method of the cleaning buffer solution comprises the following steps: and (3) measuring the volumes of the cleaning buffer solution A and the purified water according to the proportion of 1:9, adding the cleaning buffer solution A in the formula amount into the purified water, uniformly mixing, and measuring the pH value, wherein the pH value is in the range of 7.3-7.5.
Example 4
A washing buffer A: every 1000mL of wash buffer a included: 11.5g of Tris (hydroxymethyl) aminomethane (Tris), 80.0g of sodium chloride, 205.5mL of Tween-205, 9503.0mL of ProClin and the balance of purified water, wherein the pH value ranges from pH7.3 to 7.5.
A wash buffer: every 1000mL of 1 × Wash buffer contained: 100mL of washing buffer solution A and 900mL of purified water, wherein the pH value range is 7.3-7.5.
The preparation method of the washing buffer solution A comprises the following steps: weighing Tris according to a ratio, dissolving Tris with purified water, adding sodium chloride according to a formula amount, adding Tween-20 after completely dissolving, adding ProClin950 after dissolving, finally fixing the volume to a rated capacity with purified water, and adjusting the pH value of a cleaning buffer solution by using a 1M NaOH solution or an HCl solution to ensure that the pH value is within a range of pH 7.3-7.5.
The preparation method of the cleaning buffer solution comprises the following steps: and (3) measuring the volumes of the cleaning buffer solution A and the purified water according to the proportion of 1:9, adding the cleaning buffer solution A in the formula amount into pure water, uniformly mixing, and measuring the pH value, wherein the pH value is in the range of pH 7.3-7.5.
Example 5
A washing buffer A: every 1000mL of wash buffer a included: 12.2g of Tris (hydroxymethyl) aminomethane (Tris), 86g of sodium chloride, 205.0 mL of Tween-205, 9503.5 mL of ProClin and the balance of purified water, wherein the pH value ranges from pH7.3 to 7.5.
A wash buffer: every 1000mL of 1 × Wash buffer contained: 100mL of washing buffer solution A and 900mL of purified water, wherein the pH value range is 7.3-7.5.
The preparation method of the washing buffer solution A comprises the following steps: weighing Tris according to a ratio, dissolving Tris with purified water, adding sodium chloride according to a formula amount, adding Tween-20 after completely dissolving, adding ProClin950 after dissolving, finally fixing the volume to a rated capacity with purified water, and adjusting the pH value of a cleaning buffer solution by using a 1M NaOH solution or an HCl solution to ensure that the pH value is within a range of pH 7.3-7.5.
The preparation method of the cleaning buffer solution comprises the following steps: and (3) measuring the volumes of the cleaning buffer solution A and the purified water according to the proportion of 1:9, adding the cleaning buffer solution A in the formula amount into pure water, uniformly mixing, and measuring the pH value, wherein the pH value is in the range of pH 7.3-7.5.
After covering a slide cover plate on the tissue slice, dripping the cleaning solution of the embodiment 1 to the tissue slice and the cover plate, and finding that the cleaning solution can quickly and comprehensively cover the tissue and is relatively uniform; the wetting property of the cleaning solution is good; the pH of the working solution product diluted by the concentrated solution product in a ratio of 1:9 can still be maintained between 7.3 and 7.5, which indicates that the pH of the product has better stability.
The experimental results of the above examples are as follows:
table 1pH test results
| Item | Example 1 | Example 2 | Example 3 | Example 4 | Example 5 |
| pH of Wash buffer A | 7.31 | 7.42 | 7.48 | 7.37 | 7.45 |
| pH of Wash buffer | 7.32 | 7.41 | 7.49 | 7.36 | 7.46 |
TABLE 2 intensity of staining of tissue sections
Note: in table-indicates negative; + indicates a weak positive; + indicates moderate positive; + + + + indicates a strong positive.
TABLE 3 background of tissue section staining
The foregoing is only a preferred embodiment of the present application and is not intended to limit the present application, and all simple modifications, changes and equivalents of the above embodiments according to the technical spirit of the present application still belong to the protection scope of the present application.
Comparative example 1
Preparing a product by adopting the formula in the same example 1, wherein the ProClin950 is added in the example 1, and the product is a product in group A0; the other set of comparative products without ProClin950 was a1 product. The product A0 and the product A1 are respectively stored under the refrigeration condition, and the product A1 is found to be slowly turbid and polluted by microorganisms within 1-2 months, while the liquid A0 is still clear and free of microbial pollution. Similarly, the A0 product and the A1 product respectively become turbid slowly in 1-4 weeks (specifically, the cleanliness of the use environment) at room temperature, and are polluted by microorganisms; the product liquid of A0 is still clear and has no microbial contamination. The ProClin 950-containing cleaning solution of the present application enables the product to be stably preserved and used for a long period of time regardless of the refrigeration condition or room temperature condition.
Comparative example 2
A product was formulated using the formulation of example 1, wherein example 1 was a ProClin950 added as a group a0 product; the other set of comparative products was the A1 product without ProClin 950. When the reagent is used in the existing preparation, the immunohistochemical staining test is carried out on a machine: the same antibody working solution, adjacent tissue sections, the same pre-staining procedure and staining procedure were used. The dyeing results were substantially consistent. It is demonstrated that the wash solutions containing ProClin950 of the present application do not affect the binding rate of key enzymes to the substrate in the diagnostic reagent, nor do they affect the binding of antibodies.
According to the comparative example, the cleaning solution containing ProClin950 can ensure that the product can be stably stored and used for a long time no matter under the refrigeration condition or the room temperature condition; the cleaning solution of the present application does not affect the binding rate of a key enzyme to a substrate in a diagnostic reagent, nor does it affect the binding of an antibody.
Claims (10)
1. The utility model provides a washing buffer solution of full-automatic immunohistochemical staining machine adaptation which characterized in that: the washing buffer solution is formed by dissolving a prepared washing buffer solution A in purified water, wherein the main components of the washing buffer solution A comprise: tris (hydroxymethyl) aminomethane, sodium chloride, tween-20 and ProClin950, and the pH value range of the washing buffer solution A is 7.3-7.5.
2. The full-automatic immunohistochemical staining machine-adapted wash buffer of claim 1, characterized by: the volume usage ratio of the cleaning buffer solution A to the purified water is 1:9, and the pH value of the cleaning buffer solution is 7.3-7.5.
3. The full-automatic immunohistochemical staining machine-adapted wash buffer of claim 2, characterized by: the washing buffer solution A comprises the following components in each 1000 mL: 11.0-12.5 g of tris (hydroxymethyl) aminomethane), 75.0-90.0 g of sodium chloride, 203.5-6.0 mL of Tween-203, 9502.5-5.0 mL of ProClin and the balance of purified water, wherein the pH value range is pH 7.3-7.5.
4. The full-automatic immunohistochemical staining machine-adapted wash buffer of claim 3, wherein: the washing buffer solution A comprises the following components in each 1000 mL: 11.5-12.5 g of tris (hydroxymethyl) aminomethane, 80.0-90.0 g of sodium chloride, 204.5-6.0 mL of tween-204, 9503.0-5.0 mL of ProClin and the balance of purified water, wherein the pH value range is pH 7.3-7.5.
5. The full-automatic immunohistochemical staining machine-adapted wash buffer of claim 4, characterized by: the washing buffer solution A comprises the following components in each 1000 mL: 12.0-12.5 g of tris (hydroxymethyl) aminomethane, 82.0-90.0 g of sodium chloride, 205.0-6.0 mL of Tween-205, 9503.5-5.0 mL of ProClin and the balance of purified water, wherein the pH value range is pH 7.3-7.5.
6. The full-automatic immunohistochemical staining machine-adapted wash buffer of claim 5, wherein: the washing buffer solution A comprises the following components in each 1000 mL: 12.2-12.5 g of tris (hydroxymethyl) aminomethane, 85.0-90.0 g of sodium chloride, 205.5-6.0 mL of Tween-205, 9504.0-5.0 mL of ProClin and the balance of purified water, wherein the pH value range is pH 7.3-7.5.
7. A preparation method of a washing buffer solution A adapted by a full-automatic immunohistochemical staining machine is characterized by comprising the following steps: the preparation method comprises the following specific steps:
(1) weighing tris (hydroxymethyl) aminomethane according to a ratio, dissolving with purified water, adding sodium chloride according to a formula amount, adding tween-20 after completely dissolving, and adding ProClin950 after completely dissolving;
(2) fixing the volume of the solution obtained in the step (1) to a rated capacity by using purified water to obtain a cleaning buffer solution;
(3) and (3) adjusting the pH value of the washing buffer solution in the step (2) to enable the pH value to be in the range of pH 7.3-7.5, and thus obtaining the washing buffer solution A.
8. The method of preparing full-automatic immunohistochemical staining machine-adapted washing buffer a according to claim 7, characterized in that: and (3) adjusting the pH value by using a 1M NaOH solution.
9. The method of preparing full-automatic immunohistochemical staining machine-adapted washing buffer a according to claim 7, characterized in that: in the step (3), 1M HCl solution is used for adjusting the pH value.
10. A preparation method of a washing buffer solution adapted by a full-automatic immunohistochemical staining machine is characterized by comprising the following steps of: the method specifically comprises the following steps: and (3) measuring the obtained washing buffer solution A and purified water according to the volume ratio of 1:9 to obtain corresponding volumes, then adding the washing buffer solution A into pure water, uniformly mixing, and measuring the pH value within the range of pH 7.3-7.5.
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