A hematoxylin-eosin (HE) staining method without toluene and alcohol

Abstract

The present application relates to a kind of hematoxylin-eosin (HE) staining method and operation steps without xylene and alcohol, belong to pathological technique, apply in pathology, histology and clinical experimental diagnosis biomedical disciplines field such as embryo. The hematoxylin-eosin (HE) staining step of conventional paraffin section is at least 12 more additional steps before and after dyeing into dyeing liquid dyeing to mounting respectively needs to use including xylene and gradient concentration alcohol. Lengthy dyeing step is not only laborious and time-consuming, also pollutes air environment and harms personnel health. The present application is a kind of conventional paraffin section whole process hematoxylin-eosin (HE) dyeing step without using xylene and alcohol method and step, make its application in management HE dyeing process for improving pathological technical room work quality and efficiency, reduce reagent cost and eliminate environmental pollution is crucial.

Description

TECHNICAL FIELD

[0001] The present application relates to a xylene and alcohol-free hematoxylin-eosin (HE) staining method and procedure, belonging to the technical field of pathology, applied in the biomedical disciplines of pathology, histopathology and clinical laboratory diagnosis. BACKGROUND

[0002] The conventional hematoxylin-eosin staining method (referred to as HE staining method in the industry) is one of the most important techniques in morphological histopathology, and is also a key factor for pathologists to make accurate diagnosis. The existing hematoxylin-eosin (HE) staining method of paraffin sections generally includes 1. pre-staining steps: deparaffinization, 2. hematoxylin and eosin staining steps, 3. post-staining steps: dehydration and transparency, 4. mounting 4 major steps. If each operation is subdivided, there are about 25-27 specific steps in total (refer to the "Clinical Technical Operation Specification - Pathology Volume" edited by the Chinese Medical Association and published by the People's Military Medical Press, pages 23-24). As a multi-step, multi-factor determined staining method, HE staining is affected by many factors in the final staining quality and the final pathological diagnosis results, and the complexity of HE staining is not only in the technical level, but also contains some auxiliary work around HE staining. For example, the matching of equipment and staining solution, the daily inspection of the staining cylinder, the replacement of reagents during the staining process, the regular cleaning of the staining cylinder, the regularity of the section, etc. require a lot of manpower, not only time-consuming and laborious, but also pollute the air environment and harm the health of personnel.

[0003] So far, pathological industry professionals have made some innovative work to improve hematoxylin-eosin (HE) staining. There are patents focusing on improving dye solution formulations and staining methods (such as application numbers 2015109879358, 201610342055x, 2017106851082, 2017100097997, 2018103387014, 2018116127477, 201911193743, 2021101040932), and there are patents focusing on a certain step in the staining process, such as paraffin section dewaxing method (such as application number 2006101298085) and using xylene instead of reagents such as turpentine oil, castor oil, white oil and limonene and other substances to achieve paraffin section dewaxing (such as application numbers: 2016107771023, 2016107771023, 2020107828808); There are patents focusing on improving the staining kit and standardizing the staining steps (such as application numbers: 2021111628796, 2022103489936, 2022102724835). The known existing paraffin section hematoxylin-eosin (HE) staining steps require at least 12 additional steps before and after immersing in the dye solution and after staining to mount, which requires the use of xylene and alcohol of gradient concentration (Note: alcohol is commonly known as ethanol). Although few of the above patents attempt to use other organic solvents or methods to replace the xylene dewaxing step, industry users report that the effect is not very ideal, and the use of organic solvents including alcohol and the reduction of staining steps have not been substantially eliminated. The lengthy staining steps of the prior art and the accompanying liquid exchange and cleaning are the main factors leading to the above-mentioned large amount of manpower, and the volatilization of xylene and alcohol also pollutes the laboratory environment and harms the health of workers. Therefore, it is important to develop a method for hematoxylin-eosin (HE) staining of conventional paraffin sections without using xylene and alcohol or alternative reagents and short staining steps, which can be applied to manage HE staining processes to improve the quality and efficiency of pathological technology room work, reduce reagent costs, and eliminate environmental pollution. SUMMARY

[0004] In order to overcome the above-mentioned shortcomings of the prior art, the purpose of the present application is to provide a hematoxylin-eosin (HE) staining method without xylene and alcohol, which is simple to operate, environmentally friendly, safe and has good staining effect.

[0005] The technical scheme adopted by the present application to solve its technical problems is:

[0006] As a preferred embodiment, the hematoxylin-eosin (HE) staining method without xylene and alcohol, the HE staining of paraffin sections mainly includes the following steps:

[0007] (1) Immersion in Dewaxing Solution I for 3-5 minutes at a temperature range of 70-95°C;

[0008] (2) Immersion in Dewaxing Solution II for 1-5 minutes at a temperature range of 70-95°C;

[0009] (3) Immersion in Dewaxing Solution III for 1-5 minutes at a temperature range of 70-95°C;

[0010] (4) Transfer to warm water washing for 1-2 times, each for 30 seconds-1 minute at a temperature range of 40-60°C;

[0011] (5) Immersion in hematoxylin staining solution for 5-15 minutes;

[0012] (6) Water washing for 1-2 times for 1-2 minutes;

[0013] (7) Immersion in aqueous differentiation solution for 10-60 seconds;

[0014] (8) Water washing for 1-2 times, each for 1-2 minutes;

[0015] (9) Immersion in saturated aqueous lithium carbonate solution for 1-2 minutes;

[0016] (10) Water washing for 1-2 times, each for 5-10 minutes;

[0017] (11) Immersion in eosin staining solution for 20-60 seconds;

[0018] (12) Water washing for 1-2 times, each for 1-2 minutes;

[0019] (13) Transfer to hot air drying at a temperature range of 50-80°C for 5-15 minutes until the slice is completely dried;

[0020] (14) Mounting with mounting medium.

[0021] As preferred, the hematoxylin-eosin (HE) staining method without xylene and alcohol uses hematoxylin staining solution and eosin staining solution (HE) reagent components and preparation which are well-known, non-patented and conventional prior art methods.

[0022] As preferred, the dewaxing solution is a surfactant, and the surfactant includes sodium dodecyl sulfonate, sodium dodecyl sulfate, Triton X-100, Triton X-200, Triton X-405; the dewaxing solution is a water solution with a concentration of 0.5%-2% by volume or weight percentage, and is fully stirred and mixed; the total concentration of the surfactant is 0.5-2% when each is used alone or when two or more are used in combination.

[0023] As preferred, the dewaxing solution is a household detergent, including laundry detergent, laundry liquid and dishwashing detergent; the preparation method of the dewaxing solution is to dilute each single household detergent with water to form a 0.5%-2% aqueous solution, and fully stir to mix.

[0024] As preferred, the dewaxing step of the dewaxing solution I-III is reduced to two steps, which is also applicable to the dewaxing solution I-II, when the paraffin section is immersed in the dewaxing solution, 10-30% of the total volume of the dewaxing solution is added from the bottom of the dewaxing solution to flow away the surface dewaxing solution before the section is removed after reaching the time, and the surface floating mixture is eliminated, and at least the dewaxing solution I needs to add liquid to supplement.

[0025] As preferred, the aqueous differentiation solution is a mixed aqueous solution including dilute hydrochloric acid and Triton X-100 surfactant; after mixing, the concentration of the dilute hydrochloric acid is 0.5-2% and the concentration of Triton X-100 is 0.05-1%, and the surfactant is added to prevent uneven tissue differentiation on the section due to water tension, and the surfactant is any one of the above surfactants.(Note: The present application does not use the known hydrochloric acid alcohol solution differentiation).

[0026] As preferred, the water washing solution is an aqueous solution including Triton X-100 surfactant with a concentration of 0.05-1% to reduce the surface tension of water on the paraffin section slide during water washing, and ensure the consistency of tissue surface hydration on the section; the surfactant is any one of the above surfactants.

[0027] As preferred, after the section is dyed with the eosin dye solution and washed with water, the section is dried by hot air with a temperature range of 50-80°C for 5-15 minutes to replace alcohol dehydration, and the section is transparent and mounted by using mounting medium containing a transparent agent, including using xylene to prepare mounting medium to transparent the section and mount the section.

[0028] As preferred, the one hematoxylin-eosin (HE) staining method without xylene and alcohol is characterized in that, the step without indicating the temperature in the HE staining step of the paraffin section refers to room temperature, and the length of time for immersion in the dye solution and the length of time and number of times for water washing are adjusted according to environmental conditions, and the section is preferably used when it is clean and transparent.

[0029] As preferred, the HE staining step of the paraffin section has two options for implementation, which are manual operation and automatic completion by a staining machine.

[0030] Compared with the prior art, the beneficial effects of the present application are that the HE staining method without benzene and alcohol eliminates the adverse effects of xylene and alcohol organic solvents on the human body, the operation process and the staining step are short and easy to operate, no environmental pollutants are produced, the cost is low, the section staining effect is as good as that of the conventional HE staining method, and the requirements of pathological diagnosis under a microscope are met. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is an example of the staining effect under a microscope of the benzene-free and alcohol-free HE staining step in Example 1 (tumor biopsy specimen, HE staining, high magnification of the microscope).

[0032] Figure 2 The following is an example of the microscopic staining effect of the benzene-free and alcohol-free HE staining step in Table 1 of Example 2 (colonoscopic biopsy specimen, HE staining, high-magnification microscope).

[0033] Figure 3 The following is an example of the microscopic staining effect of the HE staining step of the conventional technology in Table 1 of Example 2 (colonoscopic biopsy specimen, HE staining, high magnification of the microscope).

[0034] Figure 4 This is an example of the microscopic staining effect of the benzene-free and alcohol-free HE staining step of Example 3 (ascites cancer cell paraffin block specimen, HE staining, high magnification of the microscope).

[0035] Figure 5 This is an example of the staining effect under a microscope of the benzene-free and alcohol-free HE staining step of Example 4 (fat tissue specimen, HE staining, low magnification of the microscope).

[0036] Figure 6 This is an example of the staining effect under a microscope for the benzene-free and alcohol-free HE staining step of Example 5 (tumor tissue specimen, HE staining, high magnification of the microscope). DETAILED DESCRIPTION

[0037] The reagent components and preparation of the hematoxylin and eosin (HE) staining solution used in the examples and the HE staining steps in the prior art are all well-known, traditional or prior art methods without patent protection (see "Clinical Technical Operation Specifications - Pathology Volume" pages 23-26).

[0038] The present invention is further described in conjunction with an embodiment: the xylene- and alcohol-free hematoxylin-eosin (HE) staining method for paraffin sections mainly comprises the following steps:

[0039] (1) Immerse in dewaxing solution I at a temperature range of 70-95°C for 3-5 minutes;

[0040] (2) Immerse in dewaxing solution II at a temperature range of 70-95°C for 1-5 minutes;

[0041] (3) Immerse in dewaxing solution III at a temperature range of 70-95°C for 1-5 minutes;

[0042] (4) Wash in warm water at 40-60°C 1-2 times for 30 seconds to 1 minute each time;

[0043] (5) Soak in hematoxylin stain for 5-15 minutes;

[0044] (6) Wash in water for 1-2 times, each time for 1-2 minutes;

[0045] (7) Soak in aqueous differentiation solution for 10-60 seconds;

[0046] (8) Wash in water for 1-2 times, each time for 1-2 minutes;

[0047] (9) Soak in saturated aqueous lithium carbonate for 1-2 minutes;

[0048] (10) Wash in water for 1-2 times, each time for 5-10 minutes;

[0049] (11) Soak in eosin stain for 20-60 seconds;

[0050] (12) Wash in water for 1-2 times, each time for 1-2 minutes;

[0051] (13) Move to a hot air oven at a temperature range of 50-80°C for 5-15 minutes until the section is completely dry;

[0052] (14) Mount the section with mounting medium.

[0053] The deparaffinization solution is a surfactant, which includes sodium dodecyl sulfonate, sodium dodecyl sulfate, Triton X-100, Triton X-200, Triton X-405; the deparaffinization solution is prepared by diluting each surfactant with water to a concentration of 0.5%-2% by volume or weight percentage, and fully stirring to mix.

[0054] The deparaffinization solution is a household detergent, which includes laundry detergent, laundry liquid, and dishwashing detergent; the deparaffinization solution is prepared by diluting each household detergent with water to a concentration of 0.5%-2% by volume or weight percentage, and fully stirring to mix.

[0055] The deparaffinization steps of the deparaffinization solutions I-III are reduced to two steps, which are also applicable to the deparaffinization solutions I-II, when the paraffin section is soaked in the deparaffinization solution, before being removed at the time, 10-30% of the total volume of the deparaffinization solution is added from the bottom to flow away the surface layer of the deparaffinization solution, so as to eliminate the surface floating layer of the mixture, and at least the deparaffinization solution I needs to be added.

[0056] The aqueous differentiation solution is a mixed aqueous solution comprising dilute hydrochloric acid and Triton X-100 surfactant; after mixing, the concentration of dilute hydrochloric acid is 0.5-2% and the concentration of Triton X-100 is 0.05-1%, and the surfactant is one of the above-mentioned surfactants; the surfactant is added to prevent uneven tissue differentiation on the slice due to water tension; the application does not use the known alcohol hydrochloric acid solution.

[0057] The water washing solution is an aqueous solution comprising Triton X-100 surfactant, and the concentration is 0.05-1%; the surfactant is one of the above-mentioned surfactants.

[0058] The slice is dried by hot air at a temperature range of 50-80℃ for 5-15 minutes after being dyed with the eosin dye and washed with water, to replace alcohol dehydration with dried slices, and the slice is transparent and sealed by using sealing glue containing a transparent agent, including using xylene to prepare sealing glue to make the slice transparent and sealed.

[0059] The HE staining method without xylene and alcohol is characterized in that the step without indicating the temperature in the HE staining step of the paraffin section refers to room temperature, and the length of time for immersion in the dye, the number of times of water washing and the length of time for water washing are adjusted according to environmental conditions, and the use is preferably based on the clean transparency of the slice.

[0060] The HE staining step of the paraffin section has two options, manual operation and automatic completion by a dyeing machine.

[0061] Example 1: Test the use of a whole dyeing rack (30 slices) of paraffin sections, test the dewaxing liquid, dewaxing temperature and staining step, and the benzene-free and alcohol-free HE staining step is completed by manual operation.

[0062] 1. Preparation of the dewaxing liquid: 5 milliliters of Triton X-100 (0.5% in percentage) are added to 1 liter of water, and the mixture is fully stirred and mixed to prepare; the pH value is adjusted to 7.0.

[0063] 2. Preparation of the aqueous differentiation solution: 5 milliliters of hydrochloric acid (0.5% in percentage) and 0.5 milliliters of Triton X-100 (0.05% in percentage) are added to 1 liter of water, and the mixture is fully stirred and mixed to prepare; the pH value is adjusted to 7.0.

[0064] 3. Preparation of the water washing solution: 0.5 milliliters of Triton X-100 (0.05% in percentage) are added to 1 liter of water, and the mixture is fully stirred and mixed to prepare; the pH value is adjusted to 7.0.

[0065] 4. The HE staining of the paraffin section mainly includes the following steps:

[0066] (1) Dewaxing solution I at 95°C for 3 minutes, before the slice is removed, put the liquid to supplement the liquid once (10% volume);

[0067] (2) Dewaxing solution II at 95°C for 3 minutes, before the slice is removed, put the liquid to supplement the liquid once (10% volume);

[0068] (3) Dewaxing solution III at 95°C for 3 minutes, before the slice is removed, put the liquid to supplement the liquid once (10% volume);

[0069] (4) Move into the warm water at 60°C for 1 minute;

[0070] (5) Soak into hematoxylin dye for 5 minutes;

[0071] (6) Wash with water for 1 minute;

[0072] (7) Soak into aqueous differentiation solution for 10 seconds;

[0073] (8) Wash with water for 1 minute;

[0074] (9) Soak into saturated aqueous lithium carbonate solution for 1 minute;

[0075] (10) Wash with water for 5 minutes;

[0076] (11) Soak into eosin dye for 30 seconds;

[0077] (12) Wash with water for 1 minute;

[0078] (13) Move into the hot air at 80 o C temperature for 10 minutes until the slice is completely dried;

[0079] (14) Seal the slice with mounting medium.

[0080] Example 2: Test each staining step using a whole staining rack (30) paraffin sections, test dewaxing solution, dewaxing temperature and staining steps, two staining steps are automatically completed in the same staining machine in turn.

[0081] 1. Preparation of dewaxing solution: add 10 milliliters of Triton X-100 and 10 milliliters of Triton X-405 (total percentage concentration is 2%) to 1 liter of water, mix well to prepare; adjust the pH value to 7.0.

[0082] 2. Preparation of aqueous differentiation solution: add 10 milliliters of hydrochloric acid (percentage concentration is 1%) and 5 milliliters of Triton X-100 (percentage concentration is 0.5%) to 1 liter of water, mix well to prepare; adjust the pH value to 7.0.

[0083] 3. Preparation of water washing solution: 1 milliliter of Triton X-100 (0.1% concentration) is added to 1 liter of water, and the mixture is stirred thoroughly to prepare the solution; and the pH value is adjusted to 7.0.

[0084] 4. Table 1 is the HE staining procedure without benzene and alcohol for comparison example 2 and the HE staining procedure of the known technology.

[0085]

[0086]

[0087] The staining results of the HE staining procedure without benzene and alcohol and the HE staining procedure of the known technology in Table 1 are as follows: according to the existing clinical pathology technical specification requirements, each slice is clean and transparent after being dewaxed. Under the microscope, the cell nucleus is stained blue to dark blue, the nucleolus is stained light purple red, the cell plasma, red blood cells, collagen interstitial substance, etc. are stained red to different degrees, the background is clear and the contrast is good. Figure 2 and Figure 3 are the effects of the two HE staining methods respectively, and both are determined to be qualified.

[0088] Example 3: Test using a whole staining rack (30 slices) of paraffin sections, test dewaxing solution, dewaxing temperature and staining procedure, and the HE staining procedure without benzene and alcohol is completed by manual operation.

[0089] 1. Preparation of dewaxing solution: 20 grams of sodium dodecyl sulfate (2% concentration) is added to 1 liter of water, and the mixture is stirred thoroughly to prepare the solution; and the pH value is adjusted to 7.0.

[0090] 2. Preparation of aqueous differentiation solution: 20 milliliters of hydrochloric acid (2% concentration) and 10 milliliters of Triton X-100 (1% concentration) are added to 1 liter of water, and the mixture is stirred thoroughly to prepare the solution; and the pH value is adjusted to 7.0.

[0091] 3. Preparation of water washing solution: 10 milliliters of Triton X-100 (1% concentration) is added to 1 liter of water, and the mixture is stirred thoroughly to prepare the solution; and the pH value is adjusted to 7.0.

[0092] 4. The HE staining of paraffin sections includes the following steps:

[0093] (1) Dewaxing solution I is immersed at 70°C for 5 minutes, and the solution is replaced once (30% volume) before the slice is removed;

[0094] (2) Dewaxing solution II is immersed at 70°C for 5 minutes, and the solution is ignored to be replaced;

[0095] (3) Dewaxing solution III is immersed at 70°C for 5 minutes, and the solution is ignored to be replaced;

[0096] (4) Move into 60°C warm water and wash for 2 times, 1 minute each time;

[0097] (5) Soak into hematoxylin dye solution for 15 minutes;

[0098] (6) Wash with water for 2 times, 2 minutes each time;

[0099] (7) Soak into aqueous differentiation solution for 1 minute;

[0100] (8) Wash with water for 1 time, 2 minutes;

[0101] (9) Soak into saturated aqueous lithium carbonate solution for 2 minutes;

[0102] (10) Wash with water for 1 time, 5 minutes;

[0103] (11) Soak into eosin dye solution for 1 minute;

[0104] (12) Wash with water for 2 times, 2 minutes each time;

[0105] (13) Move into 60 o C hot air for 15 minutes until the slices are completely dried;

[0106] (14) Seal the slices with mounting medium.

[0107] Example Four: Test using a whole staining rack (30 slices) of paraffin sections, test the dewaxing solution, dewaxing temperature and staining steps, benzene-free and alcohol-free HE staining steps are completed by manual operation.

[0108] 1. Preparation of the dewaxing solution: add 20 milliliters of household dishwashing detergent (2% by volume) to 1 liter of water, mix well, and adjust the pH to 7.0.

[0109] 2. Preparation of the aqueous differentiation solution: add 5 milliliters of hydrochloric acid (0.5% by volume) and 0.5 grams of sodium dodecyl sulfate (0.05% by volume) to 1 liter of water, mix well, and adjust the pH to 7.0.

[0110] 3. Preparation of the water washing solution: add 0.5 grams of sodium dodecyl sulfate (0.05% by volume) to 1 liter of water, mix well, and adjust the pH to 7.0.

[0111] 4. HE staining of paraffin sections includes the following steps (only use dewaxing solutions I and II, ignore III):

[0112] (1) Soak in dewaxing solution I at 90°C for 5 minutes, and replace the solution once (20% by volume) before removing the slices;

[0113] (2) Soak in dewaxing solution II at 90°C for 3 minutes, and replace the solution once (20% by volume) before removing the slices;

[0114] (3) Immersed in warm water at 40°C for 2 minutes for 2 times;

[0115] (4) Immersed in hematoxylin staining solution for 10 minutes;

[0116] (5) Washed with water for 2 times for 2 minutes;

[0117] (6) Immersed in aqueous differentiation solution for 40 seconds;

[0118] (7) Washed with water for 2 times for 2 minutes;

[0119] (8) Immersed in saturated aqueous lithium carbonate solution for 2 minutes for bluing;

[0120] (9) Washed with water for 2 times for 5 minutes;

[0121] (10) Immersed in eosin staining solution for 30 seconds;

[0122] (11) Washed with water for 2 times for 1 minute;

[0123] (12) Immersed in hot air at 80 o C for 10 minutes for drying until the slice is completely dried;

[0124] (13) The slice was sealed with mounting medium.

[0125] Example Five: Test using a whole staining rack (30 slices) of paraffin sections, test the dewaxing solution, benzene-free and alcohol-free HE staining steps completed by manual operation.

[0126] 1. Preparation of the dewaxing solution: 20 ml of Triton X-100 (2% concentration) was added to 1 liter of water, and fully stirred to prepare; adjust the pH value to 7.0.

[0127] 2. The HE staining steps of the paraffin sections and other reagents are the same as in Example Four.

[0128] Example Six: Test using a whole staining rack (30 slices) of paraffin sections, test the dewaxing solution, benzene-free and alcohol-free HE staining steps completed by manual operation.

[0129] 1. Preparation of the dewaxing solution: 20 grams of household laundry powder (2% concentration) was added to 1 liter of water, and fully stirred to prepare; adjust the pH value to 7.0.

[0130] 2. The HE staining steps of the paraffin sections and other reagents are the same as in Example Four.

[0131] Example Seven: Test using a whole staining rack (30 slices) of paraffin sections, test the dewaxing solution, benzene-free and alcohol-free HE staining steps completed by manual operation.

[0132] 1. Preparation of the dewaxing solution: 1 liter of water is added with 5 grams of sodium dodecyl sulfate (0.5% concentration) and mixed well; the pH value is adjusted to 7.0.

[0133] 2. The HE staining procedure of the paraffin section is the same as that of Example 4.

[0134] Example 8: A whole staining rack (30 sections) of paraffin sections is tested, the dewaxing solution is tested, and the benzene-free and alcohol-free HE staining procedure is completed by manual operation.

[0135] 1. Preparation of the dewaxing solution: 1 liter of water is added with 5 grams of household washing powder (0.5% concentration) and mixed well; the pH value is adjusted to 7.0.

[0136] 2. The HE staining procedure of the paraffin section is the same as that of Example 4.

[0137] Example 9: A whole staining rack (30 sections) of paraffin sections is tested, the dewaxing solution is tested, and the benzene-free and alcohol-free HE staining procedure is completed by manual operation.

[0138] 1. Preparation of the dewaxing solution: 1 liter of water is added with 20 milliliters of household washing agent (2% concentration) and mixed well; the pH value is adjusted to 7.0.

[0139] 2. The HE staining procedure of the paraffin section is the same as that of Example 4.

[0140] Example 10: A whole staining rack (30 sections) of paraffin sections is tested, the dewaxing solution is tested, and the benzene-free and alcohol-free HE staining procedure is completed by manual operation.

[0141] 1. Preparation of the dewaxing solution: 1 liter of water is added with 20 grams of sodium dodecyl sulfate powder (2% concentration) and mixed well; the pH value is adjusted to 7.0.

[0142] 2. The HE staining procedure of the paraffin section is the same as that of Example 4.

[0143] Evaluation of the HE staining effect of the paraffin sections in the above examples: according to the existing clinical pathology technical specification requirements, each section is dewaxed clean and transparent. Under a microscope, the cell nucleus is stained blue to dark blue, the nucleolus is stained light purple red, the cell plasma, red blood cells, and collagen interstitial are stained red to different degrees, the background is clear and has good contrast. The HE staining effect of all sections in the above examples is determined to be qualified under a microscope by a pathology professional, and only 6 microscope pictures are exemplified in this specification, as shown in Figure 1 、 Figure 2 、 Figure 3 、 Figure 4 ,Figure 5 and Figure 6 .

[0144] Table II: Comparison of the advantages and disadvantages of benzene-free and alcohol-free HE staining and existing conventional HE staining.

[0145] Comparison item No benzene and alcohol HE staining Existing conventional HE staining Reagent cost Low (surfactant) High (xylene + alcohol) Staining time Short, about 30 minutes Long, about 60 minutes Staining steps Simple, less than 15 steps of cylinder replacement Complex, about 25 steps of cylinder replacement Reagent toxicity Non-toxic substance Xylene is harmful to health Safety hazard Non-flammable reagent Flammable reagent, increasing safety risk Environmental pollution No pollution to the environment, no recycling Pollute the environment, need to be recycled Staining effect Good, meet the pathological diagnosis Good, meet the pathological diagnosis

[0146] The above merely describes preferred embodiments of the present application. Those skilled in the art should understand that the present application is not limited to the above-mentioned embodiments, and that the present application can be changed and improved without departing from the principles of the present application, and these changes and improvements should also be considered as falling within the scope of the present application.

Claims

1. A xylene- and alcohol-free hematoxylin-eosin (HE) staining method, characterized in that: HE staining of paraffin sections mainly includes the following steps: (1) Immerse in dewaxing solution I at a temperature range of 70-95°C for 3-5 minutes, (2) Immerse in dewaxing solution II at a temperature range of 70-95°C for 1-5 minutes, (3) Immerse in dewaxing solution III at a temperature range of 70-95°C for 1-5 minutes, (4) Wash in warm water at 40-60℃ for 1-2 times for 30 seconds to 1 minute each time. (5) Immerse in hematoxylin solution for 5-15 minutes. (6) Wash with water 1-2 times for 1-2 minutes. (7) Immerse in aqueous differentiation solution for 10-60 seconds. (8) Wash with water 1-2 times for 1-2 minutes each time, (9) Immerse in a saturated aqueous solution of lithium carbonate for 1-2 minutes; (10) Wash with water 1-2 times for 5-10 minutes each time, (11) Immerse in eosin solution for 20-60 seconds. (12) Wash with water 1-2 times for 1-2 minutes each time, (13) Move to 50-80℃ temperature range and dry in hot air for 5-15 minutes until the slices are completely dry. (14) Seal the slide with sealing glue; The dewaxing liquid is a surfactant, which includes sodium lauryl sulfonate, sodium lauryl sulfate, Triton X-100, Triton X-200, and Triton X-405. The dewaxing liquid is an aqueous solution prepared with water and has a concentration of 0.5% to 2% by volume or weight. When each surfactant is used alone or when two or more surfactants are mixed, the total concentration is 0.5-2%. The aqueous differentiation solution is a mixed aqueous solution comprising dilute hydrochloric acid and Triton X-100, wherein the concentration of the dilute hydrochloric acid after mixing is 0.5-2% and the concentration of Triton X-100 is 0.05-1%; The water washing solution is an aqueous solution comprising Triton X-100 with a concentration of 0.05-1%; When the paraffin section is immersed in the dewaxing solution and before the time is reached to remove it, 10-30% of the total volume of the dewaxing solution is replenished from the bottom to flow away the surface dewaxing solution and eliminate the surface floating layer mixture. At least dewaxing solution I needs to be drained and replenished.

2. The xylene- and alcohol-free hematoxylin-eosin (HE) staining method according to claim 1, wherein: The same is true for the case where the dewaxing step of the dewaxing solution I-III is reduced to a two-step dewaxing solution I-II.

3. The xylene- and alcohol-free hematoxylin-eosin (HE) staining method according to claim 1, wherein: After the sections are stained with eosin and washed with water, they are dried with hot air at a temperature range of 50-80°C for 5-15 minutes to dry the sections instead of dehydrating with alcohol; The use of mounting glue containing a transparent agent includes mounting glue prepared with xylene to transparently slice and seal the slices, combining the transparent and sealing steps into one.

4. The xylene- and alcohol-free hematoxylin-eosin (HE) staining method according to claim 1, wherein: The steps without temperature indication in the HE staining of paraffin sections refer to room temperature; the length of time of immersion in the dye solution, the number of times and duration of water washing are adjusted according to environmental conditions, and it is preferred that the sections are clean and transparent during use.

5. The xylene- and alcohol-free hematoxylin-eosin (HE) staining method according to claim 1, wherein: There are two options for performing the HE staining step of the paraffin sections: manual operation and automatic completion by a staining machine.

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