Lipid-based primary neuronal cell Golgi staining method and application

Abstract

The invention provides a lipid-based primary neuronal cell Golgi staining method and application. The method comprises the following steps: 1) extracting, culturing and fixing primary neuronal cells; 2) selecting one of lipids of phosphatidylcholine, phosphatidyl ethanolamine, sphingomyelin, cholesterol and lecithin; (3) uniformly covering the surfaces of the cells with the selected lipids, and adding Golgi staining liquid for staining; (4) removing the Golgi staining solution, and carrying out developing reaction by using a reducing agent; 5) performing optical microscope imaging observation and / or synchrotron radiation X-ray imaging observation; and 6) analyzing and processing the imaging data by using data processing software. According to the method, the primary neuronal cells are successfully dyed, the dendrites and forms of the neurons can be observed under the synchrotron radiation X-ray imaging technology, and a good research foundation is laid for quantitatively researching the complete three-dimensional structure of the neurons and the complete three-dimensional fine structure of the neurons by utilizing the synchrotron radiation X-ray.

Description

Technical Field

[0001] The invention relates to the field of neuroscience, and more specifically to a lipid-based Golgi staining method for primary neuronal cells and an application thereof. Background Art

[0002] Neuroscience, the study of the human brain, is called "the last frontier of human science". To study brain function, we must first explore the brain structure. Neurons are the basic units that make up the brain's neural network. Imaging the neuron structure will help us understand the brain's neural network. Currently, imaging of neuron structure includes imaging of neuron structure in tissues. This method obtains the structure, but when performing post-data processing, there is a disadvantage of losing structural information, or the resolution is low, and the fine structure of neurons cannot be imaged. Synchrotron radiation X-ray has the advantages of fast imaging and a large imaging field of view. It has multi-resolution imaging capabilities from low resolution to high resolution, is superior to short wavelengths, has high penetration into samples, and can achieve complete structural imaging of thick tissue samples. Therefore, synchrotron radiation X-ray imaging technology is a potential means of imaging the complete structure of neurons.

[0003] A Chinese patent application (CN202410682399.X) discloses a Golgi staining method for primary neuronal cells and its application. However, when the staining is implemented, the method introduces egg yolk impurities on the surface of the imaged cells, thereby affecting the quantitative analysis of the morphological data of the stained neurons and the image contrast under synchrotron radiation X-ray imaging. Summary of the invention

[0004] The purpose of the present invention is to provide a lipid-based Golgi staining method for primary neuronal cells and its application, so as to solve the problem that after the existing Golgi staining method is used to stain primary neuronal cells, it is impossible to use data processing software to perform quantitative analysis on the primary neuronal morphological data under an optical microscope and the image contrast under synchrotron radiation X-ray imaging.

[0005] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0006] According to a first aspect of the present invention, a lipid-based Golgi staining method for primary neuronal cells is provided, comprising the following steps: 1) extracting, culturing and fixing primary neuronal cells from animal brain tissue; 2) selecting a lipid component for staining, wherein the lipid component is selected from one of the following five types: phosphatidylcholine (PC), phosphatidylethanolamine (PE), sphingomyelin (SIM), cholesterol (Chol), and lecithin; 3) uniformly covering the surface of the primary neuronal cells with the lipid component selected in step 2), adding a Golgi staining solution to cover the surface of the primary neuronal cells after a period of reaction, and reacting, thereby staining the fixed primary neuronal cells; 4) removing the Golgi staining solution and performing a developing reaction using a reducing agent; and 5) performing optical microscopy imaging observation and / or synchrotron radiation X-ray imaging observation; 6) analyzing and processing the imaging data using data processing software.

[0007] Preferably, in step 3), the action time of the lipid component is 5 min to 60 min, and the action temperature is 10° C. to 50° C. More preferably, the action time of the lipid component is 10 min to 30 min, and the action temperature is 20° C. to 40° C. Most preferably, the action time is 15 min, and the action temperature is 26° C.

[0008] Preferably, in step 3), the concentrations of the five lipid components are: 5-10% PC, 1-5% PE, 0.1-0.5% SIM, 0.8-2.0% Chol, 5-10% lecithins. Most preferably, the concentrations of the five lipid components are: 7.02% PC, 1.58% PE, 0.23% SIM, 1.20% Chol, 9.00% lecithins.

[0009] The lipid components used in the present invention are mainly based on the main lipid components in brain tissue, namely, phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, phosphatidylcholine, and cholesterol, among which cholesterol is the most preferred.

[0010] Preferably, in step 3), the action time of the Golgi staining solution is 0.5h to 72h, and the action temperature is 4°C to -196°C. More preferably, the action time is 2h to 36h, and the action temperature is 20°C to 40°C. Optimally, the action time is 12h, and the action temperature is 26°C.

[0011] Preferably, in step 1), the fixing solution is selected from: 1% to 12.5% ​​glutaraldehyde, 2% to 10% paraformaldehyde, 5% to 10% formalin or 10% to 100% ethanol, and most preferably 2.5% glutaraldehyde.

[0012] Preferably, in step 1), the fixation time of the fixative is 10 to 60 minutes, and optimally 25 minutes.

[0013] Preferably, in step 4), the reducing agent is selected from: 10% to 30% aqueous ammonia solution or 1 to 10% aqueous lithium hydroxide solution, and most preferably 20% aqueous ammonia solution.

[0014] Preferably, in step 5), the energy of the synchrotron radiation X-ray imaging is 280 eV to 20 keV, and most preferably 525 eV.

[0015] Preferably, in step 5), the resolution of the synchrotron radiation X-ray imaging is as high as 10 nm to 1000 nm.

[0016] Preferably, in step 1), the animal brain tissue includes: SD mouse brain tissue, C57BL / 6 mouse brain tissue or BALB / c mouse brain tissue. Most preferably, it is SD mouse brain tissue (P0). It should be understood that these brain tissues are experimental brain tissues commonly used in neuroscience research. These animals are easy to obtain, reproduce quickly, have a short lifespan, and progress faster in experiments. Their brains and behaviors are similar to those of humans, so they are often used to carry out experimental studies related to neuroscience. The method of the present invention has universal applicability in these commonly used experimental brain tissues.

[0017] Preferably, in step 6), the data processing software is selected from: FijiimageJ, Graphpad Prism 8.0.1, Adobe Photoshop 2020, Amira 6.0.1, Origin 2021, Coreldraw 2020. The most preferred are FijiimageJ, Graphpad Prism 8.0.1, Adobe Photoshop 2020.

[0018] According to a second aspect of the present invention, there is provided an application of a Golgi staining method in the field of neuroscience, wherein the Golgi staining method can be used to perform Sholl analysis on primary neuron morphological data under an optical microscope using data processing software, and to perform quantitative analysis on image contrast under synchrotron radiation X-ray imaging.

[0019] It should be noted that Sholl analysis, also known as concentric circle analysis, is a quantitative analysis method for neuronal axons and dendrites, which can be used to evaluate the complexity of neurons and can be analyzed using Fiji Image J image processing software. The principle is to use the neuron cell body as the center, superimpose a set of concentric circles with the same distance on the neuron, count the number of intersections between each concentric circle and the neuron branch, and obtain the branch categories of the neuron dendrites and axons, thereby quantitatively analyzing the morphological characteristics of the neuron and reflecting the complexity of the neuron.

[0020] According to the method provided by the present invention, its working principle is: the surrounding tissue of the nerve cells in the brain tissue is a lipid-rich environment. The main lipid components in the brain tissue include PC, Chol, PE, and SIM. Lecithins complexes can also be formed with these four lipid components as the main components. The chemical structural formulas of these five lipid components are as follows: Figure 1 As shown. Golgi staining solution can stain the neuronal structure of brain tissue, but cannot stain primary neuronal cells cultured in vitro, which may be caused by the difference in the lipid environment. Based on this idea, the present invention simulates an environment similar to brain lipids by covering the surface of primary neuronal cells fixed with a fixative with a single or mixed lipid component, so that the Golgi staining solution can stain primary neuronal cells with these lipids.

[0021] The main reference basis of the lipid components used in the present invention is the main lipid components in brain tissue, i.e. phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, lecithin, and cholesterol. Among them, cholesterol is most preferably used. The final experimental results show that cholesterol has the best effect on promoting dyeing, which may be due to the fact that cholesterol has a smooth and rigid structure. When cholesterol interacts with the cell membrane, cholesterol is stuck between the cell membrane lipid molecules, which can promote the cell membrane to be more closely packed with molecules and increase the thickness of the cell membrane. It may be based on this factor, resulting in the Golgi staining solution better realizing the labeling of primary neuronal cells with the help of cholesterol.

[0022] The present invention creatively adds lipid components for the first time before Golgi staining to treat primary neuron cells, improves the staining results obtained by the existing Golgi staining method in primary neuron cell staining, successfully achieves clear observation of complete neuron structure under a microscope, and experimentally verifies that the Golgi staining method can obtain a more complete neuron structure and better imaging contrast with the assistance of cholesterol. It should be understood that the formula of the Golgi staining solution is the same as that of the prior art, and the improvement of the present invention is mainly focused on adding lipids to treat primary neuron cells before Golgi staining, using lipids to simulate a lipid environment similar to the brain during the staining process, so that the Golgi staining solution can rely on these lipids to achieve staining of primary neuron cells.

[0023] It should be noted that Chinese patent application (CN202410682399.X) discloses a Golgi staining method for primary neuronal cells and its application. Although the scheme uses egg yolk to realize the staining of primary neuronal cells using the Golgi staining method, and has good imaging contrast under synchrotron radiation X-ray imaging technology, the prior art realizes staining on the basis of adding egg yolk, which contains various biochemical components and is a complex natural substance. This leads to the use of egg yolk to assist the Golgi staining method to stain primary neuronal cells. At the same time, some impurities of egg yolk components will remain on the cell surface and around the cell. When these impurities exist in large quantities, it will lead to the inability to effectively image the structure of primary neuronal cells. When only a small amount of impurities exist, it will also affect the observation of the fine structure of neurons. A lipid-based Golgi staining method and application of primary neuronal cells proposed in the present invention solves this problem. At the same time, because the lipid components used have a clear and single chemical structure, a large amount of impurity noise will not be generated during the staining process, which will be more conducive to our quantitative analysis of primary neuronal morphological data under optical microscopy and image contrast under synchrotron radiation X-ray imaging.

[0024] Compared with the technology disclosed in the prior patent, the present invention has the following advantages: 1) The present invention further determines the promoting effect of a single component on staining, rather than being based on a natural complex, which is more conducive to understanding the mechanism of the staining optimization process from a chemical perspective (chemical structure formula). In addition, it will be of great benefit to the subsequent development and optimization of methods. For example, the effect of the concentration of a single component on staining can be further optimized in the future; 2) The sample preparation using this method is more reliable and convenient than the sample preparation method proposed in the prior art, and the staining process is less affected by other components (such as some triglycerides that are not the main components of brain lipids), and the obtained staining results produce fewer impurity artifacts.

[0025] Compared with the prior art, the positive and progressive effects of the present invention are:

[0026] 1) The staining results obtained by the Golgi staining method in primary neuronal cells are improved by adding lipids before Golgi staining. The complete neuronal structure can be clearly observed by optical microscopy imaging, and the neuronal morphology can be structurally analyzed using data processing software;

[0027] 2) Using advanced synchrotron radiation X-ray imaging technology for imaging, primary neuronal cells can be quickly and completely imaged under the same light intensity, and statistical analysis of the grayscale value of the structure can be achieved using data processing software. According to the method provided by the present invention, the complete three-dimensional structure of primary neuronal cells is imaged using synchrotron radiation X-ray imaging technology, which helps people further analyze the complete structure of neurons and can also achieve quantitative analysis of the staining effect.

[0028] 3) A lipid-based Golgi staining method for primary neuronal cells and its application proposed in the present invention solves the problem of many surface impurities and further realizes the quantitative analysis of the staining effect.

[0029] In summary, according to a lipid-based Golgi staining method for primary neuronal cells provided by the present invention, primary neuronal cells are successfully stained using a lipid-based Golgi staining method, and neuronal dendrites and morphology can be observed under synchrotron radiation X-ray imaging technology, while the neuronal morphological structure can be analyzed and the staining effect can be statistically evaluated, which lays a good research foundation for further using synchrotron radiation X-ray to study the complete three-dimensional structure of neurons and the complete three-dimensional fine structure of neurons. Such a Golgi staining method for primary neuronal cells provided by the present invention is expected to play an important role in the field of neuroscience. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is the chemical formula of five lipid components;

[0031] Figure 2A , Figure 2B , Figure 2C , Figure 2D , Figure 2E These are images of primary neuronal cells stained with Golgi tissue by adding PC, PE, SIM, Chol, and Lecithins, respectively;

[0032] Figure 3A , Figure 3B , Figure 3C , Figure 3D , Figure 3E are respectively Figure 2A , Figure 2B , Figure 2C , Figure 2D , Figure 2E An example of a single neuron obtained by extracting the neurons in the black dotted box. Figure 3F Yes Figure 2A , Figure 2B , Figure 2C , Figure 2D , Figure 2E Results The results of the sholl analysis were performed;

[0033] Figure 4A , Figure 4B , Figure 4C , Figure 4D , Figure 4E This is a synchrotron radiation X-ray image of primary neuronal cells stained with Golgi by adding PC, PE, SIM, Chol, and Lecithins. Figure 4F Yes Figure 4A , Figure 4B , Figure 4C , Figure 4D , Figure 4E Results The gray value statistical analysis results were performed. DETAILED DESCRIPTION

[0034] The present invention is further described below in conjunction with specific implementation examples. It should be understood that the following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples are unspecified specific conditions, and are selected according to conventional methods and conditions, or according to the product specifications. The reagents and raw materials used in the present invention are all commercially available.

[0035] The technical solution of the present invention mainly lies in selecting a lipid to pretreat primary neuronal cells, and proposes a Golgi staining method for synchrotron radiation X-ray imaging of primary neuronal cells. The method can be used for synchrotron radiation X-ray imaging of primary neuronal cell structures. The following examples specifically illustrate the implementation effects of the present invention.

[0036] Example 1 Five lipid components assisted Golgi staining solution to stain primary neuronal cells

[0037] SD pregnant mice were purchased from Shanghai Slake Experimental Animal Co., Ltd. When the SD pregnant mice were pregnant to the 18th day, SD mice (P0) were surgically obtained, and the brain tissue of SD mice (P0) was obtained by surgery. The brain tissue was taken out and placed in a centrifuge tube containing HBSS buffer, and then digested and degraded by trypsin and DNase to obtain a tissue suspension. After that, the enzymatic digestion and degradation was terminated using a culture medium containing MEM Eagle's with Earle's BSS, glucose, fetal bovine serum albumin, double antibody and glutamine. At this time, cells in the tube had been extracted and the cell density was diluted to 1-3×10 5 / ml. Inoculate into a culture dish containing Neurobasal medium, B-27 supplement, glutamine and double antibody culture medium for culture. Culture in vitro for at least 10 days before use in subsequent staining experiments. At 26°C, primary neuronal cells were fixed with 2.5% glutaraldehyde for 25 minutes, then the glutaraldehyde was discarded, and the cells were quickly rinsed with 1xPBS buffer to evenly cover the surface of the primary neurons with five lipid components (PC, PE, SIM, Chol, Lecithins), with the concentrations of 7.02% PC, 1.58% PE, 0.23% SIM, 1.20% Chol, and 9.00% lecithins, respectively. The cells were placed at 26°C for 15 minutes, and the prepared Golgi staining solution was added to cover the cell surface for reaction. The cells were reacted at 26°C for 12 hours, and then reduced and developed with an aqueous ammonia solution at 26°C for 10 minutes, dehydrated with gradient alcohol (30%, 50%, 75%, 90%, 95%, 100%), and finally observed under an optical microscope.

[0038] The results are as follows Figure 2A-2E As shown, all five lipid components have auxiliary effects on staining. Figure 2A This is the result of adding PC to primary neuronal cells for Golgi staining. Under the microscope, better neuronal structures can be found. Figure 2B This is the result of adding PE to primary neuronal cells for Golgi staining. Under the microscope, better neuronal structures can also be observed. Figure 2C This is the result of adding SIM to Golgi staining of primary neuronal cells. Under the microscope, a neuronal structure with relatively few dendritic branches can be observed. Figure 2D It is the result of adding Chol to Golgi staining of primary neuronal cells. Under the microscope, a neuronal structure with relatively more dendritic branches can be observed, and the neuronal structure is more complete. Figure 2E This is the result of Golgi staining of primary neuronal cells by adding Lecithins. Neuronal structures with shorter dendritic branches can be observed under a microscope.

[0039] Afterwards, we used Adobe Photoshop to extract the individual neuron structures ( Figures 3A-3E ), and combined with FijiimageJ to perform Sholl analysis on the extracted neuronal structure, such as Figure 3F As shown, using cholesterol to assist Golgi staining can obtain more complex and complete neuronal structures, that is, compared with the other four lipid components, cholesterol has the best staining-assisting effect.

[0040] Example 2 Imaging and quantitative analysis using synchrotron radiation X-ray imaging technology

[0041] SD pregnant mice were purchased from Shanghai Slake Experimental Animal Co., Ltd. When the SD pregnant mice were pregnant to the 18th day, SD mice (P0) were surgically obtained, and the brain tissue of SD mice (P0) was obtained by surgery. The brain tissue was taken out and placed in a centrifuge tube containing HBSS buffer, and then digested and degraded by trypsin and DNase to obtain a tissue suspension. After that, the enzymatic digestion and degradation was terminated using a culture medium containing MEM Eagle's with Earle's BSS, glucose, fetal bovine serum albumin, double antibody and glutamine. At this time, cells in the tube had been extracted and the cell density was diluted to 1-3×10 5 / ml. Inoculate into a culture dish containing Neurobasal mediu, B-27 supplement, glutamine and double antibody culture medium for culture. A silicon nitride window substrate is placed in this culture dish in advance. It can be used for subsequent staining experiments after at least 10 days of in vitro culture. At 26°C, use 2.5% glutaraldehyde to fix the primary neuron cells for 30 minutes, then use the glutaraldehyde to aspirate and quickly rinse with 1xPBS buffer solution, and evenly cover the surface of the primary neurons with five lipid components (PC, PE, SIM, Chol, Lecithins). After placing at 26°C for 15 minutes, add the prepared Golgi staining solution to cover the cell surface for reaction, react at 26°C for 12 hours, and then use ammonia aqueous solution for reduction development at 26°C for 10 minutes, gradient alcohol dehydration (30%, 50%, 75%, 90%, 95%, 100%), and finally use synchrotron radiation X-ray imaging technology for observation.

[0042] The experimental results are as follows Figures 4A-4E As shown in Figure 2, the five lipid components all have good imaging contrast under synchrotron radiation X-ray imaging technology, which can realize the imaging of neuronal structures. Fiji imageJ was then used to perform grayscale value statistics on the imaging results, and the results are shown in Figure 2. Figure 4F As shown in the figure, the five lipid components can all realize the statistics of grayscale values, among which Lecithins-assisted Golgi staining of primary neuronal cells has the lowest grayscale value, and Chol-assisted Golgi staining of primary neuronal cells has the highest grayscale value. This will help us further quantitatively evaluate the effect of lipid components-assisted Golgi staining of primary neuronal cells, as well as use synchrotron radiation X-ray imaging technology to perform rapid, complete, high-resolution imaging of primary neuronal cells and analyze the function of the nervous system.

[0043] The above is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. The above embodiments of the present invention can also be modified in various ways. All simple, equivalent changes and modifications made according to the claims and the contents of the specification of the present invention fall within the scope of protection of the claims of the present invention. The contents not described in detail in the present invention are all conventional technical contents.

Claims

1. A lipid-based Golgi staining method for primary neuronal cells, characterized in that: The following steps are involved: 1) Extracting primary neuronal cells from animal brain tissue and culturing the cells, and fixing the primary neuronal cells after the cells have been cultured to a certain stage; 2) selecting a lipid component for dyeing, wherein the lipid component is selected from one of the following five: phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, cholesterol, and lecithin; 3) uniformly covering the surface of the primary neuron cells with the lipid component selected in step 2), adding a Golgi staining solution to cover the surface of the primary neuron cells for reaction after a period of reaction, thereby staining the fixed primary neuron cells; 4) Remove the Golgi staining solution and use a reducing agent for development reaction; 5) Optical microscope imaging observation and / or synchrotron radiation X-ray imaging observation; and 6) Use data processing software to analyze and process the imaging data.

2. The dyeing method according to claim 1, characterized in that In step 3), the concentrations of the five lipid components are: 5-10% phosphatidylcholine, 1-5% phosphatidylethanolamine, 0.1-0.5% sphingomyelin, 0.8-2.0% cholesterol, and 5-10% phosphatidylcholine.

3. The dyeing method according to claim 1, characterized in that In step 3), the action time of the lipid component is 10 min to 30 min, and the action temperature is 20° C. to 40° C.

4. The Golgi staining method according to claim 1, wherein In step 3), the action time of the Golgi staining solution is 2h to 36h, and the action temperature is 20°C to 40°C.

5. The Golgi staining method according to claim 1, characterized in that In step 1), the fixative used to fix the primary neuronal cells is selected from: 1% to 12.5% ​​glutaraldehyde, 2% to 10% paraformaldehyde, 5% to 10% formalin or 10% to 100% ethanol; the fixation time is: 10 to 25 minutes.

6. The Golgi staining method according to claim 1, characterized in that In step 4), the reducing agent is selected from: 10% to 30% aqueous ammonia solution or 1 to 10% aqueous lithium hydroxide solution.

7. The Golgi staining method according to claim 1, characterized in that In step 5), the energy of the synchrotron radiation X-ray imaging is 280eV to 20keV, and the resolution of the synchrotron radiation X-ray imaging is 10nm to 1000nm.

8. The dyeing method according to claim 1, characterized in that The data processing software in step 6) is selected from: FijiimageJ, Graphpad Prism 8.0.1, Adobe Photoshop 2020, Amira 6.0.1, Origin 2021, Coreldraw 2020.

9. The dyeing method according to claim 8, characterized in that In step 6), Adobe Photoshop was used to extract the single neuron structure, and FijiimageJ was used to perform Sholl analysis on the extracted neuron structure to quantitatively analyze the neuron morphological characteristics and reflect the complexity of the neuron.

10. An application of the Golgi staining method for primary neuronal cells according to any one of claims 1 to 9 in the field of neuroscience, characterized in that: The Golgi staining method can realize rapid and complete three-dimensional imaging and quantitative analysis of primary neuronal cells.

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