CN106644656A - Hematoxylin-eosin one-step dyeing method - Google Patents

Abstract

The invention belongs to a routine dyeing reagent for a routine pathological section making and a use method of the routine dyeing reagent, and discloses a hematoxylin-eosin one-step dyeing method. The hematoxylin-eosin one-step dyeing method disclosed by the invention is characterized by comprising the following steps: preparing a hematoxylin-eosin one-step dyeing solution and a specific pH value buffer solution so that hematoxylin dyeing and eosin dyeing are completed in one step without the steps of differentiating and turning blue, and rinsing the sections with the specific pH value buffer solution before and after dyeing. According to the hematoxylin-eosin one-step dyeing method disclosed by the invention, one-step dyeing of cytoplasm and nucleus can be achieved, the operation steps are simplified and the operation time is shortened; the hematoxylin-eosin one-step dyeing method is good in dyeing effects and stable in results, is suitable for dyeing of routine formalin fixation and paraffin-embedded tissue sections, and can also be used for dyeing of frozen tissue sections or cultured cells, and favorable conditions are provided for rapid and accurate clinical pathological diagnosis.

Description

Hematoxylin-eosin one-step staining method

technical field

The invention relates to a hematoxylin-eosin staining solution and a staining method thereof, which belong to conventional staining reagents for routine pathological film production and methods for using the reagents.

Background technique

Hematoxylin-eosin staining (HE staining for short) is the most basic and important staining method in routine pathological slide preparation, and is widely used clinically. Hematoxylin is a basic dye that can stain the nucleus blue; eosin is an acid dye that can stain the cytoplasm red. In pathological diagnosis, teaching and scientific research, HE staining is often used to observe the morphology and structure of normal and diseased tissues. To determine or identify certain abnormal substances and special components in diseased tissues and cells, special staining methods, enzyme histochemical methods or immunohistochemical methods are required, which are also based on the observation of HE-stained tissue sections. High-quality HE-stained sections are the key to pathologists making correct diagnoses. In clinical practice, most pathological results are misdiagnosed and misjudged due to poor staining quality of sections.

Staining is one of the most important steps in the preparation of biological specimens. The biological tissue is immersed in the staining agent, so that a certain part of the tissue cells is stained with a different color or a different depth of color from other parts, resulting in different refractive indices for microscope observation. After staining, some specific solutions are used to remove the excessively combined staining agent of the tissue. This process is called differentiation, and the solution used is called differentiation solution. In conventional HE staining, 1% hydrochloric acid ethanol is commonly used as the differentiation solution, and the acid can destroy the quinone structure of hematoxylin, so that the tissue and the pigment are separated and faded. After hematoxylin staining, it must be differentiated with 1% hydrochloric acid ethanol to remove the excessively bound hematoxylin dye of the cell nucleus and the hematoxylin dye adsorbed by the cytoplasm, and then perform eosin staining to ensure that the nuclei and cytoplasm staining layers are distinct and have a sense of contrast. Hematoxylin is in a red ion state under acidic conditions and appears red; in alkaline conditions it is in a blue ion state and appears blue. After hydrochloric acid ethanol differentiation, the nuclei combined with hematoxylin dye are red, and the acid on the tissue section needs to be removed with water immediately to stop the differentiation, and then the nuclei stained with hematoxylin can be restored to blue with a weak alkaline solution. This process is called blue reversion or Blue effect.

At present, conventional HE staining adopts the method of separate staining of hematoxylin and eosin, which is not only complicated and time-consuming, but also needs to go through steps such as bluing and differentiation, which makes it difficult to grasp the degree of staining of the nucleus and cytoplasm. For example, if the staining time of hematoxylin staining solution is too long and the differentiation time is too short, the nuclei will be overstained, and the nuclear membrane and nucleolus will not be clear. ; Insufficient blue return after staining with hematoxylin staining solution, or the section stays in the hematoxylin staining solution for too short a time, or the differentiation time is too long, which can lead to unclear staining of the nucleus; too much residual blue returning solution can lead to eosin rejection, making eosin Red staining is too light or uneven; eosin staining for too long can result in a lack of contrast between nuclear and cytoplasmic staining.

All kinds of cells in the body have different sizes, shapes and functions, but their basic structure is the same. Cells are composed of nucleus, cytoplasm and cell membrane. The main component of chromatin in the nucleus is DNA. In the DNA double helix structure, the phosphate groups on the two nucleotide chains face outward, making the outside of the DNA double helix negatively charged and acidic, and it is easy to mix with the positively charged Hematoxylin basic dyes are dyed by ionic bonds or hydrogen bonds. The main chemical component of cytoplasm is protein, we know that protein has amphoteric ionization phenomenon in solution. Every protein has a pH value that can make its surface net charge zero, and this pH value is the isoelectric point of the protein. When pH>isoelectric point, the protein is negatively charged; otherwise, when pH<isoelectric point, the protein is positively charged; when pH=isoelectric point, the protein is uncharged. Since the isoelectric point of the cytoplasm is 4.7~5.0, and the pH value of the cytoplasmic environment is 6.7~6.8, which is greater than the pH value of the isoelectric point, the cytoplasmic protein appears to be negatively charged, and it may be detected by the positively charged hematoxylin basic dye. staining, resulting in indistinguishable nuclear and cytoplasmic staining.

Contents of the invention

The present invention is improved on the basis of the conventional HE staining method, and realizes one-step staining of the nucleus and cytoplasm, which not only simplifies the operation steps and shortens the operation time, but also solves the problem that the degree of staining of the nucleus and cytoplasm in conventional HE staining is difficult to grasp. .

The hematoxylin-eosin one-step dyeing solution is the root and core of the present invention, and is characterized in that: it mainly consists of hematoxylin (0.5-10%), mordant (1-5%), dyeing accelerator (0.1-1%), iodine Red Y (0.5~10%), preservative (0.01~0.1%), oxidizing agent (1~2%), distilled water and other ingredients can realize the staining of cytoplasm and nucleus in one step, the staining is uniform and clear, the sense of hierarchy is strong, and the dyeing The quality is guaranteed, which provides favorable conditions for the clinical realization of rapid and accurate pathological diagnosis.

The key to the successful implementation of the present invention is that the section is washed with a specific pH buffer solution before and after staining. The preparation of the buffer solution with a specific pH value is characterized in that: the buffer solution with a pH between 3.6 and 4.7 is prepared from a mixed solution of a weak acid and its salt or a mixed solution of a weak base and its salt. Because the isoelectric points of the nucleus and cytoplasm are different, the nucleus is about 3.3~3.6, and the cytoplasm is about 4.7~5.0. We adjust the pH value of the staining environment to be greater than the isoelectric point of the nucleus but less than the isoelectric point of the cytoplasm, that is, 3.6~ Between 4.7, at this time, the negative charge of the cell nucleus is combined with the basic dye, and the positive charge of the cytoplasm is combined with the acid dye, so that the hematoxylin contained in the one-step staining solution only stains the nucleus, and the eosin only stains the cytoplasm, and the nucleus It can be clearly distinguished from cytoplasmic staining, achieving the purpose of both successful staining and simplified staining steps.

The present invention can realize hematoxylin staining and eosin staining in one step, and omits steps such as differentiation and bluing that easily lead to staining failure, and is characterized in that the operation steps of the hematoxylin-eosin one-step staining method include:

(1) The pathological sections are routinely dewaxed before staining, and then washed with a specific pH buffer;

(2) Pathological sections were stained with hematoxylin-eosin one-step staining solution;

(3) After staining, the pathological sections were washed with a buffer solution with a specific pH value, and then subjected to routine dehydration, transparency, and sealing; (4) Judgment of staining results: the nucleus was blue, and the cytoplasm was red.

The invention is suitable for staining conventional formalin-fixed and paraffin-embedded tissue sections, and can also be used for staining frozen tissue sections or cultured cells. The tissue section processed by the present invention has a blue nucleus and a red cytoplasm, which form a sharp contrast and are easy to observe and analyze. The present invention has done staining of pathological tissue sections of human lymph nodes, uterus, gastric mucosa, large intestine, thyroid gland and other parts, and has reached the standard HE staining effect. The invention has good dyeing effect and stable result, and is a novel HE dyeing method worthy of popularization.

The invention is mainly used in hospital pathology departments, laboratory departments and scientific research laboratories, and its main components are chemical reagents without biological activity, which has good stability and can be stored at room temperature. The invention is applicable to the staining of all human and animal tissue cell slices and the popularization and use in hospitals at all levels, and provides favorable conditions for realizing rapid and accurate pathological diagnosis in clinic.

Description of drawings

Figure 1 is the result of conventional HE staining (thyroid).

Fig. 2 is the dyeing effect diagram (thyroid gland) of the present invention.

Figure 3 is the rendering of conventional HE staining (uterine wall).

Fig. 4 is a dyeing effect diagram (uterine wall) of the present invention.

detailed description

Taking conventional formalin-fixed and paraffin-embedded pathological tissue sections as an example, conventional HE staining and the operation steps of the present invention are introduced as follows.

Operation steps of conventional HE staining method.

1. Dewax the slices to water.

①Two times of xylene, 5-10 minutes each time.

②The effect of absolute ethanol is applied twice, each time for 3-5 minutes.

③ 95% ethanol for 3 to 5 minutes.

④ 90% ethanol for 3 to 5 minutes.

⑤ 80% ethanol for 3 to 5 minutes.

⑥ Rinse with tap water or distilled water for 1-3 minutes.

2. HE staining.

① Stain with modified Lillie-Mayer hematoxylin staining solution for 1-5 minutes.

②Rinse with tap water or distilled water for 10-30 seconds.

③ 1% hydrochloric acid ethanol differentiation 2 ~ 5s.

④Rinse with tap water or distilled water for 10-30 seconds.

⑤ Weak alkaline water (such as 0.2% ammonia water or 1% lithium carbonate) turns blue for 30-60 seconds.

⑥ Rinse with tap water or distilled water for 10-30 seconds.

⑦ Stain with eosin staining solution for 1-3 minutes.

⑧ Rinse with tap water or distilled water for 30-60 seconds.

3. Dehydration, transparency, and sealing.

① 80% ethanol for 10-20 seconds.

② 90% ethanol for 10-20 seconds.

③ 95% ethanol for 2 times, each time for 1 to 2 minutes.

④The effect of absolute ethanol is applied twice, each time for 2 to 3 minutes.

⑤ Xylene transparent 3 times, 2 to 3 minutes each time.

⑥ Mount with neutral resin.

4. Staining results: cell nuclei are blue; cytoplasm, muscle fibers, collagen fibers, thyroid jelly, etc. are red in different shades; keratin, red blood cells, etc. are bright orange red.

Procedure for one-step staining with hematoxylin-eosin.

1. Dewax the slices to water.

①Two times of xylene, 5-10 minutes each time.

②The effect of absolute ethanol is applied twice, each time for 3-5 minutes.

③ 95% ethanol for 3 to 5 minutes.

④ 90% ethanol for 3 to 5 minutes.

⑤ 80% ethanol for 3 to 5 minutes.

⑥Rinse with specific pH buffer solution for 30-60s.

2. One-step staining with hematoxylin-eosin (differentiation, bluing and multiple washing steps in the middle are omitted).

① Stain with hematoxylin-eosin one-step staining solution for 1-5 minutes.

②Rinse with specific pH buffer solution for 30-60s.

3. Dehydration, transparency, and mounting (the specific steps are the same as conventional HE staining).

4. The staining result is the same as conventional HE staining.

The above are only preferred embodiments of the present invention. It should be understood by those of ordinary skill in the art that the present invention is not limited by the above-mentioned embodiments. Under the premise of not departing from the principle of the present invention, the present invention can also make some changes and improvements, and these changes and improvements should also be regarded as the present invention. scope of protection.

Claims (4)

1. Hematoxylin-eosin one-step staining method, characterized in that: prepare hematoxylin-eosin one-step staining solution and specific pH value buffer solution, so that hematoxylin staining and eosin staining can be completed in one step, without differentiation and bluing steps, slices before and after staining Rinse with specific pH buffer. 2. The hematoxylin-eosin one-step staining method according to claim 1, characterized in that: the preparation of the hematoxylin-eosin one-step staining solution mainly consists of hematoxylin (0.5~10%), mordant (1~5%) , dye accelerator (0.1~1%), eosin Y (0.5~10%), preservative (0.01~0.1%), oxidant (1~2%), distilled water and other ingredients. 3. the hematoxylin-eosin one-step staining method according to claim 1, is characterized in that: the preparation of specific pH buffer solution, the pH that is configured by the mixed solution of weak acid and its salt or the mixed solution of weak base and its salt is Buffer between 3.6~4.7. 4. the hematoxylin-eosin one-step staining method according to claim 1, is characterized in that: the operation step of the hematoxylin-eosin one-step staining method comprises: (1) The pathological sections are routinely dewaxed before staining, and then washed with a specific pH buffer; (2) Pathological sections were stained with hematoxylin-eosin one-step staining solution; (3) After staining, the pathological sections were washed with a buffer solution with a specific pH value, and then subjected to routine dehydration, transparency, and sealing; (4) Judgment of staining results: the nucleus was blue, and the cytoplasm was red.