A method for golgi staining of primary neuronal cells based on lipids and application thereof

Abstract

The application provides a kind of primary neuron cell golgi staining method and application based on lipid, comprising the following steps: 1) extracting, culturing and fixing primary neuron cell;2) selecting one kind of lipid in phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, cholesterol, lecithin;3) the selected lipid is evenly covered on the cell surface, and Golgi staining solution is added for staining;4) remove Golgi staining solution, and use reducing agent for developing reaction;5) optical microscope imaging observation and / or simultaneous radiation X-ray imaging observation;6) the imaging data is analyzed and processed using data processing software.The application successfully stains primary neuron cell, and under the simultaneous radiation X-ray imaging technology, neuron dendrite and morphology can be observed, which lays a good research foundation for using simultaneous radiation X-ray to quantitatively study neuron complete three-dimensional structure and complete three-dimensional fine structure of neuron.

Description

Technical Field

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

[0002] Neuroscience, the study of the brain, has been called "the last frontier of human science." To study brain function, we must first explore brain structure. Neurons are the basic units that make up the brain's neural network, and imaging neuronal structure will facilitate our knowledge and understanding of the brain's neural network. Currently, imaging of neuronal structure involves imaging neuronal structures within tissues. This method, while obtaining structure, suffers from the drawbacks of structural information loss during post-processing, or low resolution, making it impossible to image the fine structure of neurons. Synchrotron X-rays offer the advantages of rapid imaging, a wide field of view, and multi-resolution imaging capabilities from low to high resolution. They are superior to short wavelengths, have high sample penetration, and can image the complete structure of thick tissue samples. Therefore, synchrotron 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, during the staining process, this method introduces egg yolk impurities onto the imaged cell surface, thereby affecting the quantitative analysis of the stained neuronal morphology data and 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 and application of primary neuronal cells, thereby solving 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 of primary neuronal morphological data under an optical microscope and 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: 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), reacting for a period of time, adding a Golgi staining solution to cover the surface of the primary neuronal cells for reaction, thereby staining the fixed primary neuronal cells; 4) removing the Golgi staining solution and performing a development reaction using a reducing agent; and 5) performing optical microscopy imaging observation and / or synchrotron radiation X-ray imaging observation; and 6) analyzing and processing the imaging data using data processing software.

[0007] Preferably, in step 3), the lipid component is exposed for 5 to 60 minutes at a temperature of 10° C. to 50° C. More preferably, the lipid component is exposed for 10 to 30 minutes at a temperature of 20° C. to 40° C. Most preferably, the exposure time is 15 minutes at a temperature of 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, and 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, and 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 them, cholesterol is most preferred.

[0010] Preferably, in step 3), the Golgi staining solution is incubated for 0.5 to 72 hours at a temperature of 4°C to -196°C. More preferably, the incubation time is 2 to 36 hours at a temperature of 20°C to 40°C. Optimally, the incubation time is 12 hours at a temperature of 26°C.

[0011] Preferably, in step 1), the fixative 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 most preferably 25 minutes.

[0013] Preferably, in step 4), the reducing agent is selected from: a 10% to 30% aqueous ammonia solution or a 1 to 10% aqueous lithium hydroxide solution, and most preferably a 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: Fiji imageJ, Graphpad Prism 8.0.1, Adobe Photoshop 2020, Amira 6.0.1, Origin 2021. The most preferred are Fiji imageJ, Graphpad Prism 8.0.1, and Adobe Photoshop 2020.

[0018] According to a second aspect of the present invention, a Golgi staining method for use in the field of neuroscience is provided. The Golgi staining method can be used to perform Sholl analysis on primary neuron morphological data under an optical microscope using data processing software, as well as to perform quantitative analysis of 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. It can be used to assess neuronal complexity and can be performed using Fiji Image J image processing software. The principle is to superimpose a set of concentric circles spaced at equal distances onto the neuron, with the neuron cell body as the center. The number of intersections between each concentric circle and the neuron's branches is counted to determine the branching types of the neuron's dendrites and axons, thereby quantitatively analyzing neuronal morphological characteristics and reflecting the neuron's complexity.

[0020] The working principle of the method provided by the present invention is as follows: the surrounding tissue of 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 be formed based on these four lipid components. The chemical structures 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 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, allowing the Golgi staining solution to stain primary neuronal cells with these lipids.

[0021] The main reference basis of the lipid composition adopted in the present invention is the main lipid composition in brain tissue, i.e. phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, lecithin, cholesterol. Among them, cholesterol is most preferably used. The final experimental results show that cholesterol has the best promoting effect on dyeing. This 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 tighter molecular accumulation and cell membrane thickness of the cell membrane. It may be based on this factor that the Golgi staining solution is better realized by cholesterol to the labeling of primary neuronal cells.

[0022] The present invention, for the first time, creatively adds lipid components to treat primary neuronal cells before Golgi staining, improving the staining results obtained by existing Golgi staining methods for primary neuronal cells and successfully achieving clear microscopic observation of intact neuronal structures. Furthermore, experimental verification shows that cholesterol-assisted Golgi staining can produce more complete neuronal structures and better imaging contrast. It should be understood that the formula of the Golgi staining solution is the same as that of the prior art. The improvements of the present invention primarily focus on adding lipids to treat primary neuronal cells before Golgi staining. The lipids are used to simulate a brain-like lipid environment during the staining process, allowing the Golgi staining solution to stain primary neuronal cells with these lipids.

[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 achieve the staining of primary neuronal cells using the Golgi staining method, and has good imaging contrast under synchrotron radiation X-ray imaging technology, since the prior art is based on the addition of egg yolk to achieve staining, egg yolk contains various biochemical components and is a complex natural substance. This leads to the use of egg yolk to assist in the Golgi staining method for primary neuronal cells. At the same time, some egg yolk components of impurities will remain on the cell surface and around the cell surface. When these impurities are present 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 are present, 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 component used has 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. This 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 method using this method is more reliable and convenient than the sample preparation method proposed in the prior art. 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 present invention has the following positive effects:

[0026] 1) By adding lipids before Golgi staining, the staining results obtained by the Golgi staining method in primary neuronal cells are improved. 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, primary neurons can be rapidly and completely imaged under the same light intensity. Data processing software can then be used to perform statistical analysis of the grayscale values ​​of the structures. The method provided by this invention uses synchrotron radiation X-ray imaging technology to image the complete three-dimensional structure of primary neurons, which helps further understand the complete structure of neurons and enables quantitative analysis of staining effects.

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

[0029] In summary, the lipid-based Golgi staining method for primary neuronal cells provided by the present invention successfully stains primary neuronal cells using the lipid-based Golgi staining method, and neuronal dendrites and morphology can be observed under synchrotron radiation X-ray imaging technology. At the same time, the neuronal morphological structure can be analyzed and the staining effect can be statistically evaluated, laying 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 the five lipid components;

[0031] Figure 2A 、 Figure 2B 、 Figure 2C 、 Figure 2D 、 Figure 2E These are images of primary neuronal cells stained with Golgi apparatus after addition of PC, PE, SIM, Chol, and Lecithins;

[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 conducted;

[0033] Figure 4A 、 Figure 4B 、 Figure 4C 、 Figure 4D 、 Figure 4E is the primary neuron cell synchronization radiation X-ray imaging figure of Golgi staining added with PC, PE, SIM, Chol, and lecithins respectively, Figure 4F is the primary neuron cell synchronization radiation X-ray imaging figure of Golgi staining added with PC, PE, SIM, Chol, and lecithins respectively, Figure 4A 、 Figure 4B 、 Figure 4C 、 Figure 4D 、 Figure 4E The result is subjected to gray value statistical analysis. DETAILED DESCRIPTION

[0034] The application will be further described in conjunction with specific implementation examples. It should be understood that the following examples are only used to illustrate the application and are not used to limit the scope of the application. In the following examples, the experimental methods without specific conditions are selected according to the conventional methods and conditions or according to the product instructions. The reagents and raw materials used in the application are commercially available.

[0035] The technical scheme of the application mainly lies in selecting a kind of lipid to pretreat the primary neuron cell, and a Golgi staining method for the primary neuron cell synchronization radiation X-ray imaging is proposed. The method can be used for the primary neuron cell structure synchronization radiation X-ray imaging. The following examples specifically illustrate the implementation effect of the application.

[0036] Example 1: Five kinds of lipid components respectively assist Golgi staining solution to stain primary neuron cells

[0037] The SD pregnant mice are purchased from Shanghai Slek Experimental Animal Co., Ltd. When the SD pregnant mice are pregnant to the 18th day, the SD mice (P0) are obtained by operation, and then the brain tissue of the SD mice (P0) is obtained by operation. The brain tissue is first placed in a centrifuge tube containing HBSS buffer, and then the tissue suspension is obtained by trypsin and DNase digestion and degradation. Then the digestion and degradation of the enzyme are terminated by using the medium containing MEM Eagle's with Earle's BSS, glucose, fetal bovine serum protein, double antibody and glutamine. At this time, the cells in the tube have been extracted, and the cell density is 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 aspirated and quickly rinsed with 1xPBS buffer to evenly cover the surface of primary neurons with five lipid components (PC, PE, SIM, Chol, Lecithins). The concentrations of these five components were: 7.02% PC, 1.58% PE, 0.23% SIM, 1.20% Chol, 9.00% lecithins. After standing at 26°C for 15 minutes, the prepared Golgi staining solution was added to cover the cell surface for reaction. The reaction was carried out at 26°C for 12 hours, and then ammonia aqueous solution was used for reduction development at 26°C for 10 minutes, and gradient alcohol dehydration (30%, 50%, 75%, 90%, 95%, 100%) was performed. Finally, the cells were observed under an optical microscope.

[0038] The results are as follows Figures 2A-2E As shown, all five lipid components have auxiliary effects on staining. Figure 2A This is the result of adding PC to Golgi staining of primary neuronal cells. Under the microscope, better neuronal structures can be found. Figure 2B This is the result of adding PE to the Golgi staining of primary neuronal cells. 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 perform Golgi staining on 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] Pregnant SD mice were purchased from Shanghai Slake Laboratory Animal Co., Ltd. On day 18 of gestation, SD mice were surgically harvested (P0). Brain tissue from these mice (P0) was surgically harvested and placed in a centrifuge tube containing HBSS buffer. The tissue was then digested with trypsin and DNase to obtain a tissue suspension. 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 antibodies, and glutamine. Cells were then extracted from the tube and diluted to a cell density of 1–3 × 10 5 / ml. Cells were seeded into culture dishes containing Neurobasal medium, B-27 supplement, glutamine, and double-antibody culture medium. Silicon nitride window substrates were placed in these culture dishes in advance. Cultured in vitro for at least 10 days before subsequent staining experiments. Primary neurons were fixed with 2.5% glutaraldehyde at 26°C for 30 minutes. The glutaraldehyde was then aspirated and quickly rinsed with 1x PBS buffer. Five lipid components (PC, PE, SIM, Chol, and Lecithins) were evenly coated on the surface of the primary neurons and incubated at 26°C for 15 minutes. The prepared Golgi staining solution was then added to the cell surface for reaction at 26°C for 12 hours. The cells were then reduced and developed with aqueous ammonia at 26°C for 10 minutes. Dehydration was performed using a gradient of ethanol (30%, 50%, 75%, 90%, 95%, and 100%), and finally observed using synchrotron X-ray imaging.

[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 achieve 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 data, all five lipid components can realize the statistics of grayscale values, among which Lecithins assisted Golgi staining of primary neuronal cells had the lowest grayscale value, and Chol assisted Golgi staining of primary neuronal cells had the highest grayscale value. This will help us further quantitatively evaluate the effect of lipid components assisted Golgi staining of primary neuronal cells, and use synchrotron radiation X-ray imaging technology to perform rapid, complete and high-resolution imaging of primary neuronal cells to analyze the function of the nervous system.

[0043] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of the present invention. Various modifications are possible. Any simple, equivalent changes and modifications made in accordance with the claims and description of the present invention are within the scope of protection of the patent claims. Anything not fully described in this invention is conventional technology.

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 them. After the cells reach a certain stage of culture, the primary neuronal cells are fixed; 2) selecting a lipid component for staining, wherein the lipid component is selected from one of the following five: phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, cholesterol, and phosphatidylcholine; 3) uniformly covering the surface of the primary neuronal cells with the lipid components selected in step 2), and after a period of reaction, adding a Golgi staining solution to cover the surface of the primary neuronal cells for reaction, thereby staining the fixed primary neuronal cells, wherein the effective 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; 4) Remove the Golgi staining solution and use a reducing agent for development reaction; 5) Optical microscope imaging observation and / or synchrotron 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, wherein 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.

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

4. The Golgi staining method according to claim 1, wherein 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.

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

6. The Golgi staining method according to claim 1, wherein 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.

7. An application of the Golgi staining method for primary neuronal cells according to any one of claims 1 to 6 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.

Images