A staining solution for type II alveolar cells and an in-situ specific labeling method
By providing a staining solution and in-situ specific labeling method for type II alveolar cells, using specific dye combinations and phosphotungstic acid differentiation solution, the problem of lack of high specific in-situ labeling methods in the prior art is solved, and efficient and specific labeling of type II alveolar cells is achieved, and the progress of related research has been promoted.
Patent Information
- Application Number
- CN202211012185.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-08-23
- Publication Date
- 2025-06-27
- Estimated Expiration
- 2042-08-23
AI Technical Summary
There is a lack of effective in situ specific identification methods in the prior art, and it is difficult to efficiently study and identify the morphology and adjacent relationship of type II alveolar cells.
A staining solution and in-situ specific identification method for type II alveolar cells is provided, including the use order of staining solution I, differentiation solution and staining solution II. By combining dye combinations such as acid re-red, Lichun red, orange yellow G, bright green, etc., combined with phosphotungstic acid differentiation solution, high specific identification of type II alveolar cells is achieved.
A highly specific in situ identification of type II alveolar cells is achieved, which is significantly different from background cells and can clearly display the morphology and adjacent relationship of cells, which promotes the progress of related research.
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Figure CN115436138B_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of cell labeling, and particularly relates to a staining solution for type II alveolar cells and an in-situ specific labeling method. Background Art
[0002] Alveoli are the sites for gas exchange in the lungs. They have very thin walls and are composed of single-layer type I and type II alveolar epithelia. There is a thin layer of connective tissue between adjacent alveoli, which contains elastic fibers, fibroblasts, plasma cells, macrophages, etc.
[0003] Type I alveolar cells cover about 95% of the surface area of the alveoli. Type II alveolar cells are scattered and protrude between type I alveolar cells, covering about 5% of the alveolar surface area. They are smaller in volume, cubic or round in shape, with round nuclei and lightly stained cytoplasm that appears foamy. Type II alveolar cells can synthesize and secrete a surfactant, which forms a thin film on the alveolar surface, reducing the surface tension of the alveoli and stabilizing the size of the alveoli. During breathing, when the alveoli shrink, the density of the surfactant increases, reducing the surface tension and preventing alveolar collapse; when inhaling, when the alveoli expand, the density of the surfactant decreases, increasing the alveolar retraction force and preventing alveolar overexpansion.
[0004] Type II alveolar cells are closely related to the gas exchange in the lungs and the expansion and contraction of the alveoli. Premature infants with imperfectly developed type II alveolar cells cannot produce surfactant, and the alveoli cannot expand, resulting in respiratory difficulties and even death. Modern research shows that the surfactant secreted by type II alveolar cells is an indispensable part of the innate defense system of the lungs, playing roles in aggregating and controlling pathogens, regulating the functions of macrophages, and inhibiting virus infections.
[0005] The relationship between the morphology and function of type II alveolar cells is a research hotspot in life sciences. There are 8,736 relevant literature reports that can be found only on CNKI, covering aspects such as the proliferation, atrophy, necrosis, expression of active substances, and functional roles of active substances in type II alveolar cells. However, due to the lack of in-situ labeling methods for alveolar cells, existing research on type II alveolar cells mostly uses methods such as in-vitro cultured cells, exfoliated cells obtained by bronchoalveolar lavage, or detection of related active substances in lung tissue homogenates, and there are fewer studies on morphology and adjacent relationships using in-situ methods. Regarding the research on pathological morphological damages such as pulmonary fibrosis, alveolar effusion, hyperplasia or metaplasia of alveolar epithelial cells caused by COVID-19 infection, there are 28 reports in the Chinese Journal of Pathology alone, and many of them mention the pathological changes of type II alveolar cells. With the continuous in-depth research, type II alveolar cells, which play an important role in innate defense and immune regulation, are one of the indispensable research objects, but there is a lack of existing in-situ specific labeling methods.
[0006] Existing in-situ labeling and research methods for type II alveolar cells:
[0007] 1. HE staining method (Figure 1 ) Hematoxylin and eosin (HE) staining is widely used in the routine morphological observation of various tissue cells. This method can stain the cytoplasm of cells pink and the cell nuclei blue. It does not have the ability to specifically identify cell types. When observing cells using this method, cell types are mostly identified based on the morphological characteristics of the cells, and specific identification cannot be carried out, which is not conducive to large-area observation.
[0008] 2. Electron microscopy method ( Figure 2 ) The electron microscopy method can clearly show alveolar cells and various cells in the pulmonary interstitium, and it is the gold standard for morphological identification; Cui Guangbin, Wei Jingguo, etc. [The value of ruthenium red staining in the study of type II alveolar epithelial cells, Journal of the Fourth Military Medical University [J], 2002(23)03]; By adding ruthenium red to the pre-fixative of the electron microscopy fixative, a clearer structural diagram of type II alveolar cells was obtained. However, the electron microscopy method is affected by the sampling area and can only show individual cells, and it is impossible to observe a large area to obtain overall lesion information. Coupled with the cumbersome specimen preparation process and high detection cost, it is not easy to become a routine method for detecting a large number of specimens.
[0009] 3. Immunohistochemistry method ( Figures 3-5 ) Yao Lin, Zhang Junwei, etc. [The effect of Qinbai Qingfei Concentrated Pills on the repair of type II alveolar epithelial cells in rats infected with Mycoplasma pneumoniae. China Journal of Traditional Chinese Medicine [J], 2016(46)08;] used immunohistochemistry to detect surfactant in situ on the surface of type II alveolar cells in rats. The alveolar cells and pulmonary interstitium were both shown as brown, without showing specificity; Surfactant protein C (SP-C) is considered a specific marker substance for type II alveolar cells. Some researchers [The effect of long-term intragastric administration of aflatoxin G1 on the expression of SP-C and PCNA in alveolar epithelium of NIH mice, Shen Haitao, Zhang Xianghong, etc. Cancer Prevention and Treatment Research, 2007(34)5] used immunohistochemistry to label SP-C in type II alveolar cells of neonatal rats and obtained good results. However, positive expression was also visible in other cells of the alveolar wall, and specificity was not well reflected; Xiong Yi, Huang Zhongxin, etc. used [The expression and significance of surfactant protein-B in type II alveolar cells, Anatomical Research, 2002(24)4] immunohistochemistry to label SP-b in type II alveolar cells of human fetal lungs and obtained specific identification.
[0010] 4. Alkaline phosphatase (AKP)-Nuclear fast red staining method ( Figure 6 ) Jiang Haiying, Zeng Qingfu, etc. [A simple staining method for type II alveolar epithelial cells. Jiang Haiying, Zeng Qingfu, Chinese Journal of Clinical and Experimental Pathology, 1999(05)03] used alkaline phosphatase (AKP)-nuclear fast red to label exfoliated type II alveolar cells obtained by bronchoalveolar lavage. Few cells were obtained, and both type II alveolar cells and neutrophils showed positive reactions, without showing high specificity; This method can be seen in other literature for reference applications.
[0011] 5. Flow cytometer detection of in vitro cultured cells Jia Mingwang and Huang Wenjie used TUNEL fluorescence to label apoptotic type II alveolar cells cultured in vitro and used flow cytometry to detect the labeled cells. Although the cells were directly detected, the cell morphology and the adjacent relationship with the surrounding tissues could not be observed, and it could only provide apoptosis information of alveolar cells.
[0012] 6. In vitro cell culture method for detecting active substances: Gao Jing, Du Qingbo and others used IL-31 to act on type II alveolar cells cultured in vitro to study the effect on type II alveolar cell chemokine CC ligand 2; Gao Xiaowei and Zou Xinzhong used type II alveolar cells obtained by digestion and separation of rat lung tissue for in vitro culture to study the effect and mechanism of tobacco extract on the expression of TGF / smld2 signaling pathway in type II alveolar cells, etc.
[0013] A review of existing literature reports on type II alveolar cells shows that the main research is to detect active substances or cell morphology by in vitro cultured cells, or to obtain a small amount of exfoliated type II alveolar cells by bronchial lavage for morphological observation; there are few in situ cell morphology and adjacent relationships or active substance detection. Although HE staining and electron microscopy can be used for in situ morphological observation, they have limitations, and immunohistochemistry is not easy to obtain high specificity. Questions about what methods can be used to specifically identify type II alveolar cells can also be seen on the Dingxiangyuan APP. The applicant has not been able to easily retrieve methods for in situ specific identification of type II alveolar cells through the search of existing literature; the in situ morphology of type II alveolar cells, as well as its intuitive morphological and functional relationship research such as proliferation, reduction, atrophy, hypertrophy, necrosis, metaplasia and adjacent relationships, need to establish a highly specific in situ specific identification method to promote the progress of type II alveolar cell related research. Summary of the invention
[0014] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a staining solution for type II alveolar cells and an in situ specific labeling method.
[0015] The object of the present invention is achieved through the following technical scheme: a staining solution for type II alveolar cells, the staining solution comprising staining solution I, staining solution II and differentiation solution, the use order of the staining solution is to use staining solution I first, then differentiation solution, and finally staining solution II;
[0016] The dye solution I comprises the following components by weight: 0.1-2 parts of acid fuchsin, 0.1-4 parts of ponceau, 0.1-3 parts of orange G, and 200-500 parts of 0.1-0.25% acetic acid solution;
[0017] The dyeing solution II comprises the following components by weight: 0.1 to 1 part of brilliant green, 50 to 300 parts of 0.1 to 0.25% acetic acid solution;
[0018] The differentiation solution is a 3-8% phosphotungstic acid solution.
[0019] Furthermore, the staining solution I, by weight, comprises the following components: 1 part of acid fuchsin, 2 parts of ponceau, 2 parts of orange G, and 300 parts of 0.2% acetic acid solution.
[0020] Furthermore, the staining solution II, by weight, comprises the following components: 0.5 part of brilliant green and 150 parts of 0.2% acetic acid solution.
[0021] Furthermore, the differentiation solution is a 5% phosphotungstic acid solution.
[0022] The present invention also provides a staining solution for type II alveolar cells and an in-situ specific labeling method, comprising the following steps:
[0023] S1. Dewax the section to water and stain it with the staining solution I according to claim 1 for 0.2-5 minutes; preferably 1 minute.
[0024] S2. Wash the excess staining solution I on the section, then differentiate it with the differentiation solution according to claim 1 for 5-15 minutes, preferably 10 minutes, and then wash the excess differentiation solution; specifically, the differentiation time is judged according to the differentiation effect observed under the microscope. For example, when it is seen that except for type II alveolar cells, the other background cells are lightly colored or not colored, it is judged that the differentiation is completed and the next step can be carried out.
[0025] S3. Stain it with the staining solution II according to claim 1 for 8-30 minutes, preferably 10 minutes.
[0026] S4. After washing, dehydrate, clear, and then mount the section.
[0027] Furthermore, in steps S2 and S4, the solution used for washing is: acetic acid solution.
[0028] Furthermore, in steps S2 and S4, the number of washings is two.
[0029] Furthermore, in steps S2 and S4, the concentration of the acetic acid solution is 0.2%.
[0030] Furthermore, in step S4, the method of dehydration is to soak it with absolute ethanol for dehydration;
[0031] And / or, in step S4, the method of clearing is to use one of the reagents such as xylene, biological section clearing agent, and chloroform for clearing; it should be noted that the reagent used for clearing needs to have the property of being miscible with absolute ethanol and dissolving the resin glue, and other conventional reagents for clearing can be used under the condition of meeting the above conditions.
[0032] And / or, in step S4, the method of mounting the coverslip is to use neutral balsam for mounting.
[0033] Furthermore, in step S1, the sections are fixed with a weakly acidic fixative or a neutral fixative before use. The weakly acidic fixative refers to a fixative with a pH higher than 5.0, such as formalin prepared with tap water, 95% ethanol, etc.
[0034] Furthermore, in step S1, the sections are obtained from lung tissue fixed with formalin or neutral formalin before use.
[0035] It should be noted that in the present invention, the operations and reagents related to cleaning, dehydration, clearing, mounting, etc. can all be adaptively adjusted in combination with the existing technology.
[0036] The beneficial effect of the present invention is that the present invention discloses an in-situ labeling method for type II alveolar cells, and the labeled type II alveolar cells have high specificity. Description of the Drawings
[0037] Figure 1 In-situ labeling of type II alveolar cells by HE staining method; among them, the cytoplasm of rat lung tissue cells is stained pink and the cell nuclei are stained blue, and type II alveolar cells are not specifically shown (20×)
[0038] Figure 2 In-situ labeling of type II alveolar cells by electron microscopy method; among them, the electron microscopy method can clearly show local individual type II alveolar cells
[0039] Figure 3 In-situ labeling of type II alveolar cells by alkaline phosphatase-nuclear fast red method; among them, exfoliated type II alveolar cells and neutrophils in bronchoalveolar lavage rats are all stained (100×)
[0040] Figure 4 In-situ labeling of type II alveolar cells by immunohistochemistry method (SP-a); among them, SP-a can be seen expressed in rat alveolar wall cells, and the specificity is poor (20×)
[0041] Figure 5 In-situ labeling of type II alveolar cells by immunohistochemistry method (SP-C); among them, SP-C can be seen expressed in neonatal rat alveolar wall cells, and the specificity is poor (20×)
[0042] Figure 6 In-situ labeling of type II alveolar cells by immunohistochemistry method (SP-b); among them, SP-b is specifically expressed in human fetal lung type II alveolar cells (40×)
[0043] Figure 7The in-situ specific labeling method for type II alveolar cells in Example 1; wherein, the cytoplasm of type II alveolar cells is labeled pink and the cell nuclei are blue, which are significantly different from the background color (20×).
[0044] Figure 8 is Figure 7 The partial enlarged view; wherein, it can be clearly seen that the labeled type II alveolar cells are highly specific;
[0045] Figure 9 The in-situ specific labeling method for type II alveolar cells in Example 2. Specific implementation mode
[0046] The technical solution of the present invention will be further described in detail below with reference to the drawings, but the protection scope of the present invention is not limited to the following description.
[0047] Example 1
[0048] Experimental object: Rat lung tissue
[0049] Experimental reagents:
[0050] Staining solution No. 1: 1 g of acid fuchsin, 2 g of ponceau, and 2 g of orange G are dissolved in 300 ml of 0.2% acetic acid aqueous solution;
[0051] Staining solution No. 2: 0.1 g of light green is dissolved in 100 ml of 0.2% acetic acid aqueous solution;
[0052] Acetic acid washing solution: 0.2 ml of glacial acetic acid is dissolved in 100 ml of distilled water;
[0053] Phosphotungstic acid aqueous solution: 5 g of phosphotungstic acid is dissolved in 100 ml of distilled water;
[0054] Absolute ethanol;
[0055] Xylene;
[0056] Neutral gum mounting agent.
[0057] Experimental method: Take the animal specimens fixed with formalin, dehydrate with gradient ethanol, make transparent with xylene, infiltrate with wax, and prepare continuous sections of 3 μm after paraffin embedding. The sections are dewaxed to water, stained with staining solution No. 1 for 1 minute, washed twice with acetic acid aqueous solution, differentiated with phosphotungstic acid aqueous solution for 10 minutes, washed twice with acetic acid aqueous solution, stained with staining solution No. 2 for 10 minutes, washed twice with acetic acid aqueous solution, dehydrated with absolute ethanol, made transparent with xylene, and mounted with neutral gum.
[0058] Experimental results: The experimental results are shown in Figure 7 and Figure 8 , from Figure 7 , 8It can be clearly seen that the cytoplasm of type II alveolar cells is marked pink and the nuclei are blue, significantly different from the background color, and it can be clearly seen that the marked type II alveolar cells are highly specific.
[0059] Example 2
[0060] Experimental subjects: Mouse lung tissue
[0061] Experimental reagents:
[0062] Staining solution No. 1: 0.5 g of acid fuchsin, 1 g of ponceau, and 1 g of orange G are dissolved in 150 ml of 0.2% acetic acid aqueous solution;
[0063] Staining solution No. 2: 0.5 g of light green is dissolved in 300 ml of 0.2% acetic acid aqueous solution;
[0064] Acetic acid washing solution: 0.2 ml of glacial acetic acid is dissolved in 100 ml of distilled water;
[0065] Phosphotungstic acid aqueous solution: 5 g of phosphotungstic acid is dissolved in 100 ml of distilled water;
[0066] Absolute ethanol;
[0067] TO biological section clearing agent;
[0068] Neutral balsam mounting medium.
[0069] Experimental method: Take mouse lung tissue fixed with neutral formalin, dehydrate with gradient ethanol, clear with TO biological section clearing agent, impregnate with wax, and prepare continuous 3-μm sections after paraffin embedding. Dewax the sections to water, drop-stain with Staining solution No. 1 for 1 minute, wash twice with acetic acid aqueous solution, differentiate with phosphotungstic acid aqueous solution for 10 minutes, wash twice with acetic acid aqueous solution, drop-stain with Staining solution No. 2 for 10 minutes, wash twice with acetic acid aqueous solution, dehydrate with absolute ethanol, clear with TO biological section clearing agent, and mount with neutral balsam.
[0070] Experimental results: The experimental results are shown in Figure 9 . From Figure 9 It can be clearly seen that the cytoplasm of type II alveolar cells is marked pink and the nuclei are blue, significantly different from the background color, and it can be clearly seen that the marked type II alveolar cells are highly specific.
[0071] The above are only the preferred embodiments of the present invention. It should be understood that the present invention is not limited to the forms disclosed herein, should not be regarded as excluding other embodiments, but can be used in various other combinations, modifications and environments, and can be changed within the scope of the concept described herein through the above teachings or the techniques or knowledge in the relevant fields. And the changes and modifications made by those skilled in the art without departing from the spirit and scope of the present invention should all be within the protection scope of the appended claims of the present invention.
Claims
1. A staining solution for type II alveolar cells, characterized in that, The staining solution includes Staining Solution I, Staining Solution II, and differentiation solution. The using sequence of the staining solution is to first use Staining Solution I, then use the differentiation solution, and finally use Staining Solution II; The Staining Solution I, by weight, includes the following components: 0.1 - 2 parts of acid fuchsin, 0.1 - 4 parts of ponceau, 0.1 - 3 parts of orange G, 200 - 500 parts of 0.1 - 0.25% acetic acid solution; The Staining Solution II, by weight, includes the following components: 0.1 - 1 part of light green, 50 - 300 parts of 0.1 - 0.25% acetic acid solution; The differentiation solution is 3 - 8% phosphotungstic acid solution.
2. The staining solution for type II alveolar cells according to claim 1, wherein The Staining Solution I, by weight, includes the following components: 1 part of acid fuchsin, 2 parts of ponceau, 2 parts of orange G, 300 parts of 0.2% acetic acid solution.
3. A staining solution for type II alveolar cells according to claim 1, wherein, The Staining Solution II, by weight, includes the following components: 0.5 part of light green, 150 parts of 0.2% acetic acid solution.
4. A staining solution for type II alveolar cells according to claim 1, characterized in that, The differentiation solution is 5% phosphotungstic acid solution.
5. An in-situ specific labeling method for type II alveolar cells, characterized in that, It includes the following steps: S1. Dewax the section to water and stain it with the Staining Solution I described in Claim 1 for 0.2 - 5 minutes; S2. Wash the excess Staining Solution I on the section, then differentiate it with the differentiation solution described in Claim 1 for 5 - 15 minutes, and then wash the excess differentiation solution; S3. Stain it with the Staining Solution II described in Claim 1 for 8 - 30 minutes; S4. After washing, dehydrate, clear, and then mount the section; Among them, in step S1, the section is fixed with weak acidic formalin or neutral formalin before use.
6. The in-situ specific labeling method for type II alveolar cells according to claim 5, characterized in that, In steps S2 and S4, the solution used for washing is: acetic acid solution.
7. The in-situ specific labeling method for type II alveolar cells according to claim 5, characterized in that, In steps S2 and S4, the number of washings is two.
8. The in-situ specific labeling method for type II alveolar cells according to claim 6, characterized in that, In steps S2 and S4, the concentration of the acetic acid solution is 0.2%.
9. The in-situ specific labeling method for type II alveolar cells according to claim 5, characterized in that, In step S4, the method of dehydration is to soak it with absolute ethanol for dehydration; and / or, in step S4, the method of clearing is to clear it with one of the reagents of xylene, biological section clearing agent, and chloroform; and / or, in step S4, the method of mounting is to mount it with neutral balsam.