CN111413289B - Method for monitoring oxidation degree of hematoxylin staining solution and method for controlling quality of hematoxylin staining solution - Google Patents
Method for monitoring oxidation degree of hematoxylin staining solution and method for controlling quality of hematoxylin staining solution Download PDFInfo
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Abstract
The invention relates to a method for monitoring the oxidation degree of hematoxylin staining solution and a method for controlling the quality of the hematoxylin staining solution, belonging to the technical field of hematoxylin staining solution. The method for monitoring the oxidation degree of the hematoxylin staining solution comprises the following steps: (1) detecting the OD value of the diluent of the hematoxylin staining solution before oxidation by using an ultraviolet spectrophotometer and recording the OD value as OD0(ii) a (2) Detecting the OD value of the dilution of the oxidized hematoxylin staining solution by using an ultraviolet spectrophotometer and recording the OD value as ODt(ii) a (3) Before and after oxidation, the OD value rule of the diluent of the hematoxylin staining solution is changed as follows: the greater the degree of oxidation, the greater the value of the OD value; according to ODtRelative to OD0The degree of oxidation of the hematoxylin staining solution was monitored. The quality control method is rapid and accurate, and is easy to popularize and use. The method solves the problem that the quality of the existing hematoxylin staining solution cannot be controlled, so that the stained slices are good in quality and bad in time.
Description
Technical Field
The invention relates to a method for monitoring the oxidation degree of hematoxylin staining solution and a method for controlling the quality of the hematoxylin staining solution, belonging to the technical field of hematoxylin staining solution.
Background
Hematoxylin-Eosin staining method (HE staining method for short) is the most common staining method in each hospital laboratory and the most classical staining technique in each pathology department. The HE staining method is based on the principle that chromatin in cell nuclei is mainly composed of deoxyribonucleic acid (abbreviated as DNA), and in the double helix structure of DNA, phosphate groups on both strands are outward, negatively charged and acidic, and easily bind to positively charged hematoxylin basic dye in an ionic bond or hydrogen bond manner to stain, and hematoxylin is blue in an alkaline solution, so that the cell nuclei are stained blue. The results shown after hematoxylin staining were: the nucleus was blue, the cartilage matrix and calcium salt particles dark blue, and the mucus grayish blue. The cytoplasm is dyed by the cytoplasm dye eosin, so that various components of the cytoplasm show pink colors with different depths. After staining by hematoxylin-eosin staining method, various tissues or cell components and general morphological structure characteristics of pathological changes can be shown.
The core of the HE staining method is the preparation of hematoxylin staining solution, which is the key for determining the quality of the staining. The hematoxylin staining solution has a plurality of formulas, the principle is the same, the hematoxylin is firstly oxidized, one hydroxyl in an oxidized molecule becomes a carbonyl group to form a conjugated pi bond, so that the oxidized hematoxylin has the staining capability; if oxidation continues, hematoxylin peroxide is formed, the residual hydroxyl groups are further destroyed by the oxidative conjugated pi bonds, and the chromophore is destroyed and loses the staining ability. The hematoxylin oxide is combined with nuclear chromatin under the action of mordant. Two methods of oxidation of hematoxylin are commonly used: firstly, natural oxidation and secondly, artificial oxidation. However, both oxidation methods are not easy to grasp the oxidation degree, if the oxidation degree is not enough, the coloration is slow and the dyeing power is weak, if the oxidation is excessive, particle precipitation and a surface metal oxide film are easily formed, the dyeing effect and the service life are influenced, the oxidation degree of the hematoxylin dyeing solution used influences the quality of dyed slices, and if the oxidation degree of the hematoxylin dyeing solution cannot be confirmed, the quality of the dyed slices is good and bad.
In order to improve the quality of the stained slices, a staining solution containing hematoxylin with controllable quality needs to be obtained, and at present, the main strategies adopted are as follows: one strategy is to adjust the dyeing time frequently and remove the metal oxide film, and the other strategy is to prepare a new HE dyeing solution; both of these two methods are cumbersome and affect the working efficiency.
Disclosure of Invention
The invention aims to provide a method for monitoring the oxidation degree of hematoxylin staining solution, which relieves the condition that the oxidation degree of the hematoxylin staining solution cannot be confirmed in the prior art, so that dyed slices are good in quality and bad in time.
The second purpose of the invention is to provide a quality control method of hematoxylin staining solution.
The technical scheme of the invention is as follows:
a method for monitoring the oxidation degree of hematoxylin staining solution comprises the following steps:
(1) detecting the OD value of the diluent of the hematoxylin staining solution before oxidation by using an ultraviolet spectrophotometer and recording the OD value as OD0；
(2) Detecting the OD value of the dilution of the oxidized hematoxylin staining solution by using an ultraviolet spectrophotometer and recording the OD value as ODt；
(3) Before and after oxidation, the OD value rule of the diluent of the hematoxylin staining solution is changed as follows: the greater the degree of oxidation, the greater the value of the OD value; according to ODtRelative to OD0The degree of oxidation of the hematoxylin staining solution was monitored.
The method for monitoring the oxidation degree of the hematoxylin staining solution can be carried out according to ODtRelative to OD0The degree of increase of the hematoxylin staining solution is monitored, and the monitoring method is simple to operate, clear in result and easy to implement. The condition that the oxidation degree of hematoxylin staining solution can not be confirmed in the prior art, so that stained slices are good in quality and bad in time is relieved.
It should be understood that the hematoxylin staining solution prior to oxidation means that the hematoxylin staining solution that has not been oxidized either naturally or by a peroxide; the oxidized hematoxylin staining solution refers to oxidized hematoxylin staining solution.
It will be appreciated that the concentration of hematoxylin in the dilutions used in steps (1) and (2) is identical.
The principle of the method for monitoring the oxidation degree of the hematoxylin staining solution is as follows: before being oxidized, hematoxylin staining solution has OD value under the detection of ultraviolet spectrophotometer, but after being oxidized by oxidant, one hydroxyl in the molecule is oxidized into carbonyl to form conjugatePi bond increases the staining ability of oxidized hematoxylin, increases the ultraviolet absorption OD value, and monitors the oxidation degree of hematoxylin by detecting the OD value after oxidation. The oxidized hematoxylin is further mediated by ions (such as Al)3+Or is Fe3+) Further staining of the cell nuclei.
In order to further improve the monitoring accuracy and accuracy, the detection wavelength of the ultraviolet spectrophotometer is preferably 562 nm. When the detection wavelength of the ultraviolet spectrophotometer is 562nm, the difference of the OD values of the hematoxylin staining solution before and after oxidation is obvious, and the accuracy and precision of monitoring the oxidation degree are improved.
In order to further improve the monitoring accuracy and precision, the diluted hematoxylin staining solution is preferably obtained by diluting the hematoxylin staining solution with 5% aluminum sulfate aqueous solution. The 5% aluminum sulfate aqueous solution has a small influence on the OD value, and the difference in OD value between the diluted solutions of hematoxylin staining solutions before and after oxidation can be greatly increased by diluting the hematoxylin staining solution with the 5% aluminum sulfate aqueous solution as a diluent.
Preferably, the concentration of hematoxylin in the dilution of hematoxylin staining solution is 0.125 mg/mL. When the concentration of the hematoxylin in the diluent of the hematoxylin staining solution is 0.125mg/mL, and the OD value of the hematoxylin after oxidation is in the range of 2.5-3.5, the oxidation degree is proper, the hematoxylin staining solution before oxidation is only 0.23, namely, the OD value difference of the hematoxylin before and after oxidation is large.
The specific type of hematoxylin staining solution is not limited, and hematoxylin staining solutions conventional in the art may be used, such as Ehrlich hematoxylin staining solution, Harris hematoxylin staining solution, Cole hematoxylin staining solution, Mayer hematoxylin staining solution, Carazzi hematoxylin staining solution, modified lilie-Mayer hematoxylin staining solution, and Gill modified hematoxylin staining solution.
Preferably, the hematoxylin staining solution is mainly prepared by the following method: dissolving hematoxylin and aluminum sulfate in water, adding sodium iodate for oxidation, separating to remove solids, collecting separated liquid, adding glycerol, and mixing to obtain hematoxylin staining solution.
Preferably, the mass ratio of the hematoxylin to the aluminum sulfate is 1: 10. The mass ratio of the hematoxylin to the sodium iodate is 10: 1. The temperature of the oxidation was 45 ℃. The oxidation time is 2-5 min. The volume fraction of the glycerol in the hematoxylin staining solution is 5%.
A quality control method of hematoxylin staining solution comprises the following steps:
(1) monitoring the oxidation degree of the hematoxylin staining solution by adopting the method for monitoring the oxidation degree of the hematoxylin staining solution, and evaluating the oxidation degree of the hematoxylin staining solution by using an OD value; determining qualified OD value by combining the dyeing effect of hematoxylin dyeing liquid, and recording as ODs(ii) a The OD value is obtained by detecting a diluent of hematoxylin staining solution;
(2) detecting the OD value of the diluent of the hematoxylin staining solution to be detected by using an ultraviolet spectrophotometer and recording the OD value as ODtDetermine ODtWhether or not OD is satisfieds(ii) a If the condition is met, the quality of the hematoxylin staining solution to be detected is qualified.
The quality control method of hematoxylin staining solution of the invention only needs to determine ODsAnd then, quickly determining whether the quality of the hematoxylin staining solution to be detected is qualified according to the OD value of the dilution of the hematoxylin staining solution to be detected, so that the hematoxylin is oxidized to a proper degree, and the staining effect is greatly improved. The quality control method is rapid and accurate, and is easy to popularize and use. The method solves the problem that the quality of the existing hematoxylin staining solution cannot be controlled, so that the stained slices are good in quality and bad in time.
In order to improve the accuracy and precision of the quality control of the hematoxylin staining solution, the detection wavelength of the ultraviolet spectrophotometer is preferably 562 nm.
In order to improve the accuracy and precision of the quality control of the hematoxylin staining solution, the hematoxylin staining solution is preferably diluted by 5% aluminum sulfate water solution.
In order to improve the accuracy and precision of the quality control of the hematoxylin staining solution, the concentration of hematoxylin in the dilution of the hematoxylin staining solution is preferably 0.125 mg/mL.
Preferably, the hematoxylin staining solution is mainly prepared by the following method: dissolving hematoxylin and aluminum sulfate in water, adding sodium iodate for oxidation, separating to remove solids, collecting separated liquid, adding glycerol, and mixing to obtain hematoxylin staining solution; diluting the hematoxylin staining solution by using 5% of aluminum sulfate water solution until the concentration of hematoxylin is 0.125mg/mL to form a dilution solution of the hematoxylin staining solution; using 562nm as the detection wavelength of an ultraviolet spectrophotometer; if the OD value of the diluent of the hematoxylin staining solution to be detected is 2.5-3.5, the quality of the hematoxylin staining solution is qualified; if the OD value of the diluent of the hematoxylin staining solution to be detected is less than 2.5, the oxidation degree is not enough, and the quality of the hematoxylin staining solution can be qualified by continuing oxidation; and if the OD value of the diluent of the hematoxylin staining solution to be detected is greater than 3.5, over-oxidizing, and determining that the quality of the hematoxylin staining solution is unqualified.
It should be understood that when the oxidation degree is not enough, the hematoxylin staining solution can reach a more ideal OD value by continuing oxidation, so that the quality of the hematoxylin staining solution is qualified; the oxidation method is not limited, and oxidation methods conventional in the art, such as adding an oxidizing agent, increasing the oxidation time, increasing the oxidation temperature, and the like, may be used. When the hematoxylin is high in oxidation degree and excessively oxidized, the hematoxylin can be directly discarded without use, and the dyed slices are effectively prevented from being poor in quality.
Drawings
FIG. 1 is a 40-fold microscopic view of spleen after staining by the HE staining procedure in test example 2;
FIG. 2 is a graph showing the staining effect of test example 3 with OD values of 1.05, 4.63, and 2.53.
Detailed Description
The present invention will be further described with reference to the following embodiments.
In the embodiment of the invention, the preparation method of the hematoxylin staining solution before oxidation comprises the following steps: fully dissolving the same raw materials as the hematoxylin basic staining solution, and mixing to obtain the hematoxylin basic staining solution. It should be understood that, in order to avoid oxidation of the hematoxylin staining solution before oxidation, the detection should be performed quickly after the completion of the preparation of the hematoxylin staining solution before oxidation.
In the examples of the present invention, the basic preparation method of hematoxylin staining solution (100mL) used was:
(1) 0.5g of hematoxylin and 5g of aluminum sulfate are weighed, put into a beaker, added with 90ml of purified water for dissolution, and heated in a water bath kettle to promote dissolution.
(2) When the solution is completely dissolved and the temperature of the solution is raised to 45 ℃, 0.05g of sodium iodate is added, the temperature is kept for 2min, and the stirring is not stopped, so that the full oxidation is ensured.
(3) The tap water was cooled to room temperature and filtered through filter paper.
(4) Adding 5mL of glycerol protective reagent (volume fraction is about 5%), and mixing uniformly.
In the examples of the present invention, a method of preparing hematoxylin staining solutions (100mL) using different amounts of oxidizing agents was used:
(1) weighing 0.5g of hematoxylin and 5g of aluminum sulfate, putting the hematoxylin and the aluminum sulfate into a beaker, adding 90ml of purified water for dissolution, and heating in a water bath kettle to promote dissolution;
(2) when the solution is completely dissolved and the temperature of the solution is raised to 45 ℃, adding different amounts of sodium iodate (shown in table 1), preserving the temperature for 2min, and keeping stirring to ensure full oxidation;
(3) cooling tap water to room temperature, and filtering with filter paper;
(4) adding 5mL of glycerol protective reagent (volume fraction is about 5%), and mixing uniformly.
TABLE 1 amount of oxidizing agent sodium iodate
| 1 | 2 | 3 | 4 | 5 | |
| Amount of oxidizing agent | 0.001g | 0.025g | 0.05g | 0.10g | 0.20g |
In the examples of the present invention, the method for preparing hematoxylin staining solution (100mL) by oxidizing with the oxidizing agent for different periods of time:
(1) 0.5g of hematoxylin and 5g of aluminum sulfate are weighed, put into a beaker, added with 90ml of purified water for dissolution, and heated in a water bath kettle to promote dissolution.
(2) When the dissolution was complete and the temperature of the solution was raised to 45 ℃, 0.05g of sodium iodate was added and the oxidation was continued for various periods of time (as shown in table 2) with no agitation to ensure adequate oxidation.
(3) The tap water was cooled to room temperature and filtered through filter paper.
(4) Adding 5mL of glycerol protective reagent (volume fraction is about 5%), and mixing uniformly.
TABLE 2 time of oxidation
| 1 | 2 | 3 | 4 | 5 | 6 | |
| Time of oxidation | 30s | 2min | 5min | 20min | 40min | 60min |
In the embodiment of the present invention, HE staining procedure is adopted to determine the staining effect of hematoxylin staining solution, and may be a conventional HE staining procedure in the art, or may be an HE staining procedure as in test example 2.
The specific embodiment of the method for monitoring the oxidation degree of the hematoxylin staining solution comprises the following steps:
example 1
In the method for monitoring the oxidation degree of the hematoxylin staining solution of the present embodiment, 562nm is used as the detection wavelength of the ultraviolet spectrophotometer, the OD values of the dilutions of the hematoxylin staining solution corresponding to different oxidant dosages as shown in table 1 are detected, and the oxidation degree is determined, wherein the dilutions of the hematoxylin staining solution used for detection are obtained by diluting the hematoxylin staining solution with 5% aluminum sulfate aqueous solution, and the concentration of hematoxylin in the dilutions of the hematoxylin staining solution is 0.125 mg/mL; the method comprises the following steps:
(1) detecting the OD value of the diluent of the hematoxylin staining solution before oxidation by using an ultraviolet spectrophotometer and recording the OD value as OD0， OD0＝0.23。
(2) Detecting the OD value of the dilution of the oxidized hematoxylin staining solution by using an ultraviolet spectrophotometer and recording the OD value as ODtAs shown in table 3, the OD values of the dilutions of the hematoxylin staining solution at different amounts of the oxidizing agent were 0.58, 1.76, 2.53, 4.56, and 4.63, respectively.
(3) Before and after oxidation, the OD value rule of the diluent of the hematoxylin staining solution is changed as follows: the greater the degree of oxidation, the greater the value of the OD value; according to ODtRelative to OD0The degree of oxidation of the hematoxylin staining solution was monitored.
TABLE 3 amount of sodium iodate as oxidant and the corresponding OD value
| 1 | 2 | 3 | 4 | 5 | |
| Amount of oxidizing agent | 0.001g | 0.025g | 0.05g | 0.10g | 0.20g |
| ODt | 0.58 | 1.76 | 2.53 | 4.56 | 4.63 |
As can be seen from Table 3, the numbers 1 to 5, ODtRelative to OD0The increase degree becomes larger, according to the monitoring method of the embodiment, the following can be obtained: the oxidation degree of the hematoxylin staining solution increased from number 1 to number 5. This is consistent with the fact that the oxidation degree is larger when the amount of the oxidant is larger, which proves that the method for monitoring the oxidation degree of the hematoxylin staining solution of the present embodiment is feasible and can accurately judge the relative high or low of the oxidation degree.
Example 2
In the method for monitoring the oxidation degree of the hematoxylin staining solution of the present embodiment, 562nm is used as the detection wavelength of the ultraviolet spectrophotometer, the OD values of the dilutions of the hematoxylin staining solution corresponding to different oxidation times shown in table 2 are detected, the oxidation degree is determined, the dilution of the hematoxylin staining solution used for detection is obtained by diluting the hematoxylin staining solution with 5% aluminum sulfate aqueous solution, and the concentration of hematoxylin in the dilution of the hematoxylin staining solution is 0.125 mg/mL; the method comprises the following steps:
(1) detecting the OD value of the diluent of the hematoxylin staining solution before oxidation by using an ultraviolet spectrophotometer and recording the OD value as OD0， OD0＝0.23。
(2) Detecting the OD value of the dilution of the oxidized hematoxylin staining solution by using an ultraviolet spectrophotometer and recording the OD value as ODtAs shown in table 4, the OD values of the dilutions of the hematoxylin staining solution at different oxidation times were 1.05, 2.98, 3.31, 4.56, 4.99, and 5.7, respectively.
(3) Before and after oxidation, the OD value rule of the diluent of the hematoxylin staining solution is changed as follows: the greater the degree of oxidation, the greater the value of the OD value; according to ODtRelative to OD0The degree of oxidation of the hematoxylin staining solution was monitored.
TABLE 4 Oxidation differences and corresponding OD values
| 1 | 2 | 3 | 4 | 5 | 6 | |
| Time of oxidation | 30s | 2min | 5min | 20min | 40min | 60min |
| ODt | 1.05 | 2.98 | 3.31 | 4.56 | 4.99 | 5.7 |
As can be seen from Table 4, from the sequence No. 1 toNumber 6, ODtRelative to OD0The increase degree becomes larger, according to the monitoring method of the embodiment, the following can be obtained: the oxidation degree of the hematoxylin staining solution increased from number 1 to number 6. This is consistent with the increase of the oxidation time and the larger the oxidation degree, which proves that the method for monitoring the oxidation degree of the hematoxylin staining solution of the embodiment is feasible and can accurately judge the relative high or low of the oxidation degree.
Example 3
In the method for monitoring the oxidation degree of the hematoxylin staining solution of the present embodiment, 562nm is used as the detection wavelength of the ultraviolet spectrophotometer, the dilution of the hematoxylin staining solution used for detection is obtained by diluting the hematoxylin staining solution with 5% aluminum sulfate aqueous solution, and the concentration of hematoxylin in the dilution of the hematoxylin staining solution is 0.125 mg/mL; the method comprises the following steps:
(1) detecting the OD value of the diluent of the hematoxylin staining solution before oxidation by using an ultraviolet spectrophotometer and recording the OD value as OD0。
(2) Detecting the OD value of the dilution of the oxidized hematoxylin staining solution by using an ultraviolet spectrophotometer and recording the OD value as ODt。
(3) Before and after oxidation, the OD value rule of the diluent of the hematoxylin staining solution is changed as follows: the greater the degree of oxidation, the greater the value of the OD value; according to ODtRelative to OD0The degree of oxidation of the hematoxylin staining solution was monitored. ODtRelative to OD0The greater the increase in the amount of the hematoxylin staining solution, the higher the degree of oxidation.
Can be determined according to the OD of the dilution of two hematoxylin staining solutions to be detectedtRelative to OD0The relative oxidation degree, OD, of the two hematoxylin staining solutions to be detected is judgedtRelative to OD0The degree of increase is large and the degree of oxidation is relatively large.
Secondly, the specific embodiment of the quality control method of the hematoxylin staining solution of the invention is as follows:
example 4
The quality control method of the hematoxylin staining solution of the embodiment comprises the following steps:
(1) monitoring the oxidation degree of the hematoxylin staining solution by using the method for monitoring the oxidation degree of the hematoxylin staining solution in the embodiment 1, and evaluating the oxidation degree of the hematoxylin staining solution according to the OD value; combined with the staining effect of hematoxylin staining solution, as ODtWhen the dyeing effect is 2.53, the dyeing effect is best, and the OD value with qualified quality is determined and recorded as ODs(2.53); the OD value is obtained by detecting a diluent of hematoxylin staining solution.
(2) Detecting the OD value of the diluent of the hematoxylin staining solution to be detected by using an ultraviolet spectrophotometer and recording the OD value as ODtDetermine ODtWhether or not OD is satisfieds(2.53); if the condition is met, the quality of the hematoxylin staining solution to be detected is qualified.
Example 5
The quality control method of the hematoxylin staining solution of the embodiment comprises the following steps:
(1) monitoring the oxidation degree of the hematoxylin staining solution by using the method for monitoring the oxidation degree of the hematoxylin staining solution in the embodiment 2, and evaluating the oxidation degree of the hematoxylin staining solution according to the OD value; combined with the staining effect of hematoxylin staining solution, as ODtAt 2.98 and 3.31, the dyeing effect was best, and the OD values which were qualified in quality were determined and recorded as ODs(2.98-3.31); the OD value is obtained by detecting a diluent of hematoxylin staining solution.
(2) Detecting the OD value of the diluent of the hematoxylin staining solution to be detected by using an ultraviolet spectrophotometer and recording the OD value as ODtDetermine ODtWhether or not OD is satisfieds(2.98-3.31); if the condition is met, the quality of the hematoxylin staining solution to be detected is qualified.
Example 6
In the quality control method of the hematoxylin staining solution of the present embodiment, 562nm is used as the detection wavelength of the ultraviolet spectrophotometer, and the dilution of the hematoxylin staining solution used for detection is obtained by diluting the hematoxylin staining solution with 5% aluminum sulfate aqueous solution, and the concentration of hematoxylin in the dilution of the hematoxylin staining solution is 0.125 mg/mL; the method comprises the following steps:
(1) the degree of oxidation of the hematoxylin staining solution was monitored by the method for monitoring the degree of oxidation of the hematoxylin staining solution of example 3, and evaluated by OD valueOxidation degree of the hematoxylin staining solution; combined with the staining effect of hematoxylin staining solution, as ODtHas a dyeing ability of 1.00 or more, when OD istWhen the oxidation degree of the hematoxylin staining solution is between 2.5 and 3.5, the oxidation degree of the hematoxylin staining solution is proper, the staining quality is high, the staining quantity is large, and the hematoxylin staining solution is easy to store.
(2) Detecting the OD value of the diluent of the hematoxylin staining solution to be detected by using an ultraviolet spectrophotometer and recording the OD value as ODtDetermine ODtWhether or not OD is satisfieds(2.5-3.5); if the OD value of the diluent of the hematoxylin staining solution to be detected is 2.5-3.5, the quality of the hematoxylin staining solution is qualified; if the OD value of the diluent of the hematoxylin staining solution to be detected is less than 2.5, the oxidation degree is not enough, and the quality of the hematoxylin staining solution can be qualified by continuing oxidation; and if the OD value of the diluent of the hematoxylin staining solution to be detected is greater than 3.5, over-oxidizing, and determining that the quality of the hematoxylin staining solution is unqualified.
Third, related test example
Test example 1 wavelength of ultraviolet spectrophotometer
Taking hematoxylin staining solution obtained by hematoxylin staining solution basic preparation method (oxidation temperature is 45 deg.C, oxidation time is 2min) as detection object, and obtaining appropriate detection wavelength of ultraviolet spectrophotometer.
The basic formulation of hematoxylin staining solution is shown in table 5.
TABLE 5 basic recipe composition of hematoxylin staining solution
| Hematoxylin staining solution | 100mL |
| Hematoxylin | 0.5g |
| Aluminium sulphate | 5g |
| Sodium iodate | 0.05g |
| Glycerol | 5mL |
The basic preparation method of the hematoxylin staining solution comprises the following steps:
(1) 0.5g of hematoxylin and 5g of aluminum sulfate are weighed, put into a beaker, added with 90ml of purified water for dissolution, and heated in a water bath kettle to promote dissolution.
(2) When the solution is completely dissolved and the temperature of the solution is raised to 45 ℃, 0.05g of sodium iodate is added, the temperature is kept for 2min, and the stirring is not stopped, so that the full oxidation is ensured.
(3) The tap water was cooled to room temperature and filtered through filter paper.
(4) Adding 5mL of glycerol protective reagent (volume fraction is about 5%), and mixing uniformly.
50 μ L of the hematoxylin staining solution was sampled, diluted to 2mL with 5% aluminum sulfate aqueous solution, and the mixture was mixed to obtain a diluted solution of oxidized hematoxylin staining solution, and the OD values obtained by detecting the diluted solution of oxidized hematoxylin staining solution are shown in table 6.
Meanwhile, each component in the hematoxylin was tested, i.e., 2mL of each of purified water as component 1, 5% (volume fraction) aluminum sulfate aqueous solution as component 2, 5% (volume fraction) glycerin aqueous solution as component 3, and 0.05% (volume fraction) sodium iodate aqueous solution as component 4 was directly tested from each of the above four components, and the obtained OD values were as shown in table 6. The raw materials of the non-oxidized hematoxylin solution are shown in table 5, after the preparation, 50 μ L of the sample is immediately taken under the condition of maintaining non-oxidation, the sample is diluted to 2mL by using 5% aluminum sulfate aqueous solution, and the mixture is uniformly mixed and then detected, so that the OD value corresponding to the dilution of the non-oxidized hematoxylin solution is obtained.
TABLE 6 OD values at different detection wavelengths
As can be seen from table 6, when the detection wavelength is 562nm, the change of the OD value of the oxidized hematoxylin compared with the OD value of the hematoxylin before oxidation is obvious, and when the detection wavelength is 562nm, the influence of other components (purified water, aluminum sulfate and glycerol) on the OD value is small, and water and aluminum sulfate have no absorption to the ultraviolet light with the wavelength of 562nm, and have no influence on the detection result. When the detection wavelength is 433nm, 540nm and 595nm, the change of the OD value of the hematoxylin is not obvious before and after oxidation, so the detection wavelength of the ultraviolet spectrophotometer is preferably 562 nm.
Test example 2HE staining procedure
In the quality control method of the hematoxylin staining solution, the judgment of the staining effect of the hematoxylin staining solution is judged by using the staining effect of the HE staining program, the spleen is stained according to the HE staining program shown in the table 7, and the obtained stained spleen 40-fold mirror is shown in figure 1, so that the staining effect is good, and the staining quality is high.
TABLE 7HE staining procedure
The preparation method of the hematoxylin staining solution (500mL) comprises the following steps: 1) weighing 2.5g of hematoxylin, and putting the hematoxylin into a beaker; (2) weighing 75g of aluminum sulfate, pouring into a liquid preparation pot, adding 400mL of purified water, and heating for dissolving; (3) when the reagent is completely dissolved and the temperature of the solution is raised to 45 ℃, 0.25g of sodium iodate is added, the temperature is kept for 2min, and stirring is not stopped, so that sufficient oxidation is ensured; (3) cooling tap water to room temperature; (4) filtering with filter paper; (7) adding 25mL of glycerol protective reagent (volume fraction is about 5%), and mixing uniformly.
The preparation method of the differentiation solution (500mL) comprises the following steps: 20g of tartaric acid is weighed, 490mL of purified water is added, the mixture is stirred by a stirrer to be fully dissolved, and the pH value is adjusted to 2.4-2.6 by 5M NaOH solution.
The preparation method of the bluing solution (500mL) comprises the following steps: weighing 7.5g of Tris alkali and 5g of sodium chloride, pouring the Tris alkali and the sodium chloride into a measuring cup/liquid preparation barrel, adding 490mL of purified water, stirring by using a stirrer to fully dissolve, adding ProClin950 reagent (liquid biological preservative), fully mixing uniformly and adjusting the pH value of the solution to 7.5-7.7.
The preparation method of eosin dye solution (500mL) comprises the following steps: weighing 5g of water-soluble eosin Y, adding 250mL (about 47.5%) of 95% alcohol and 150mL (30%) of ethylene glycol, stirring to dissolve completely, adjusting pH to about 4.5, and adding 100mL of purified water.
Test example 3
In order to visually judge the dyeing effect mentioned in example 6, the dyeing effect corresponding to the more typical OD values (OD values of 1.05, 4.63, and 2.53) of example 6 was characterized by using 2.53 having an OD of 2.5 to 3.5 as a typical example, 1.05 having an OD of less than 2.5 as a typical example, and 4.63 having an OD of less than 3.5 as a typical example, by the HE dyeing program of test example 2, and the obtained results are shown in fig. 2.
As can be seen from FIG. 2, the dyeing ability was exhibited at OD of 1.05 to 4.63, but it is clear that the dyeing quality was high at OD of 2.53, and that the dyeing ability was weak due to oxidation of the hematoxylin portion at OD of 1.05, resulting in prolonged dyeing time, too light dyeing, and decreased dyeing amount. When the OD is 4.63, the hematoxylin is excessively oxidized, a metal oxide film is easily generated on the prepared hematoxylin due to excessive oxidation, the excessively oxidized hematoxylin loses the dyeing capability, the dyeing quality is reduced, the dyeing quantity is also obviously reduced, the placing time of the hematoxylin dyeing liquid with the OD of 4.63 is not easy to overlong, the prepared dyeing liquid needs to be quickly used up in a short time, the dyeing quantity is reduced due to slight delay, only the unused dyeing liquid is discarded, waste is caused, and the cost is increased.
Claims (8)
1. A method for monitoring the oxidation degree of hematoxylin staining solution is characterized by comprising the following steps:
(1) detecting the OD value of the diluent of the hematoxylin staining solution before oxidation by using an ultraviolet spectrophotometer and recording the OD value as OD0；
(2) Detecting the OD value of the dilution of the oxidized hematoxylin staining solution by using an ultraviolet spectrophotometer and recording the OD value as ODt；
(3) Before and after oxidation, the OD value rule of the diluent of the hematoxylin staining solution is changed as follows: the greater the degree of oxidation, the greater the value of the OD value; according to ODtRelative to OD0Monitoring the degree of oxidation of the hematoxylin staining solution;
the detection wavelength of the ultraviolet spectrophotometer is 562 nm.
2. The method of monitoring the degree of oxidation of a hematoxylin staining solution according to claim 1, wherein the diluted hematoxylin staining solution is obtained by diluting the hematoxylin staining solution with 5% aluminum sulfate aqueous solution.
3. The method for monitoring the degree of oxidation of a hematoxylin staining solution according to claim 1, wherein the concentration of hematoxylin in the dilution of the hematoxylin staining solution is 0.125 mg/mL.
4. The method for monitoring the degree of oxidation of a hematoxylin staining solution according to any one of claims 1 to 3, wherein the hematoxylin staining solution is prepared by a method comprising: dissolving hematoxylin and aluminum sulfate in water, adding sodium iodate for oxidation, separating to remove solids, collecting separated liquid, adding glycerol, and mixing to obtain hematoxylin staining solution.
5. A quality control method of hematoxylin staining solution is characterized by comprising the following steps:
(1) monitoring the oxidation degree of the hematoxylin staining solution by using the method for monitoring the oxidation degree of the hematoxylin staining solution according to any one of claims 1 to 3, and evaluating the oxidation degree of the hematoxylin staining solution by using an OD value; determining that the OD value range of qualified quality is 2.5-3.5 by combining the dyeing effect of the hematoxylin dyeing liquid; the OD value is obtained by detecting a diluent of hematoxylin staining solution;
(2) detecting the OD value of the diluent of the hematoxylin staining solution to be detected by using an ultraviolet spectrophotometer and recording the OD value as ODtDetermine ODtWhether the concentration is between 2.5 and 3.5; if so, determining that the quality of the hematoxylin staining solution to be detected is qualified;
the detection wavelength of the ultraviolet spectrophotometer is 562 nm.
6. The method for controlling the quality of a hematoxylin staining solution according to claim 5, wherein the dilution of the hematoxylin staining solution is obtained by diluting the hematoxylin staining solution with 5% aluminum sulfate aqueous solution.
7. The method for controlling the quality of a hematoxylin staining solution according to claim 5, wherein the concentration of hematoxylin in the diluted solution of the hematoxylin staining solution is 0.125 mg/mL.
8. The method for controlling the quality of the hematoxylin staining solution according to any one of claims 6 to 7, wherein the hematoxylin staining solution is prepared by the following method: dissolving hematoxylin and aluminum sulfate in water, adding sodium iodate for oxidation, separating to remove solids, collecting separated liquid, adding glycerol, and mixing to obtain hematoxylin staining solution;
if the OD value of the diluent of the hematoxylin staining solution to be detected is 2.5-3.5, the quality of the hematoxylin staining solution is qualified; if the OD value of the diluent of the hematoxylin staining solution to be detected is less than 2.5, the oxidation degree is not enough, and the quality of the hematoxylin staining solution can be qualified by continuing oxidation; and if the OD value of the diluent of the hematoxylin staining solution to be detected is greater than 3.5, over-oxidizing, and determining that the quality of the hematoxylin staining solution is unqualified.
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