A Golgi staining method for primary neurons and its application

Abstract

This invention provides a Golgi staining method for primary neurons and its application, comprising the following steps: 1) extracting and culturing primary neurons; 2) fixing the primary neurons with a fixative; 3) uniformly covering the cell surface with egg yolk, and after a period of time, removing the egg yolk, adding Golgi staining solution, and staining the fixed primary neurons; 4) removing the Golgi staining solution and reacting with a reducing agent; and 5) optical microscopy imaging and / or synchrotron X-ray imaging observation. This invention successfully stains primary neurons, and neuronal dendrites and morphology can be observed under synchrotron X-ray imaging technology, laying a good research foundation for further research on the complete three-dimensional structure and fine three-dimensional structure of neurons using synchrotron X-ray, which will promote human understanding of information communication in the nervous system.

Description

Technical Field

[0001] This invention relates to the field of neuroscience, and more specifically to a Golgi staining method for primary neurons and its application. Background Technology

[0002] Neuroscience, the study of the human brain, is often referred to as "the last frontier of human science." To study brain function, the first step is to explore its structure. Neurons are the basic units that make up the brain's neural networks, and imaging their structure is crucial for understanding these networks. Current methods for imaging neuronal structures include imaging neurons within tissues. However, this method suffers from drawbacks during post-processing, such as loss of structural information or low resolution, failing to capture the fine details of neurons. Other methods involve imaging the complete three-dimensional structure of neurons. These require transfection or specific labeling of neurons to achieve fluorescent labeling of the structure, followed by imaging using fluorescence microscopy or super-resolution fluorescence microscopy. However, fluorescence microscopy cannot simultaneously provide rapid, wide-field-of-view, high-resolution imaging of the entire neuronal structure. Furthermore, fluorescence signals undergo photoquenching under prolonged illumination, leading to loss of structural information and increased difficulty in sample preservation.

[0003] Because synchrotron X-ray imaging offers advantages such as speed, a large field of view, and multi-resolution imaging capabilities from low to high resolution, it is a potential method for imaging neuronal structures. Currently, for staining or labeling primary neurons, apart from transfection or specific antibody labeling, no other staining or labeling method can label neuronal structures and simultaneously be used for synchrotron X-ray imaging. In tissues, Golgi staining has achieved good staining and labeling of neuronal structures, and it can also image the stained neuronal structures under synchrotron X-ray imaging. However, at the cellular level, Golgi staining has not yet yielded effective structural staining results. Nevertheless, based on the "mercuryophilicity" of neurons, it is still possible to achieve effective staining of primary neurons by optimizing the Golgi staining method, and this method can produce good imaging results under synchrotron X-ray imaging. Summary of the Invention

[0004] The purpose of this invention is to provide a Golgi staining method for primary neurons and its application, thereby solving the problems that existing Golgi staining methods cannot stain primary neurons and that synchrotron radiation X-ray imaging technology cannot perform high-resolution complete three-dimensional structural imaging of primary neurons.

[0005] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0006] According to a first aspect of the present invention, a method for Golgi staining of primary neurons is provided, comprising the following steps: 1) extracting and culturing primary neurons from animal brain tissue; 2) fixing the primary neurons using a fixative; 3) uniformly covering the surface of the primary neurons with egg yolk, reacting for a period of time, removing the egg yolk, adding a prepared Golgi staining solution to cover the surface of the primary neurons for further reaction, thereby staining the fixed primary neurons; 4) removing the Golgi staining solution and reacting with a reducing agent; and 5) observing the stain using optical microscopy and / or synchrotron radiation X-ray imaging.

[0007] Preferably, in step 3), the egg yolk is reacted for 5 to 60 minutes at a temperature of 10°C to 30°C. More preferably, the egg yolk is reacted for 10 to 30 minutes at a temperature of 20°C to 30°C. Most preferably, the reacting time is 15 minutes at a temperature of 26°C.

[0008] Preferably, in step 3), the Golgi staining solution is applied for 0.5 h to 72 h at a temperature of 4 °C to 80 °C. More preferably, the application time is 6 h to 72 h at a temperature of 10 °C to 30 °C. Most preferably, the application time is 12 h at a temperature of 26 °C.

[0009] Preferably, in step 2), the fixative is selected from: 1%–12.5% ​​glutaraldehyde, 2%–10% paraformaldehyde, 5%–10% formalin, or 10%–100% ethanol. Most preferably, it is 4% paraformaldehyde.

[0010] Preferably, in step 2), the fixation time of the fixative is 10–60 min. Most preferably, it is 30 min.

[0011] 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. Most preferably, it is a 20% aqueous ammonia solution.

[0012] Preferably, in step 5), the energy of the synchrotron radiation X-ray imaging is 280 eV to 20 keV. Most preferably, it is 525 eV.

[0013] Preferably, in step 5), the resolution of the synchrotron radiation X-ray imaging is as high as 10nm to 1000nm.

[0014] 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 (PO). It should be understood that these brain tissues are commonly used experimental brain tissues in neuroscience research. These animals are readily available, reproduce rapidly, have short lifespans, allow for faster experimental progress, and their brains and behaviors are similar to humans, thus they are frequently used to conduct neuroscience-related experimental research. The method of this invention is universally applicable to these commonly used experimental brain tissues.

[0015] According to a second aspect of the present invention, an application of the Golgi staining method in the field of neuroscience is provided, wherein the Golgi staining method can achieve rapid, high-resolution, and complete three-dimensional imaging of primary neuronal cells, including optical microscopy imaging and synchrotron radiation X-ray imaging.

[0016] According to the method provided by the present invention, the working principle is as follows: egg yolk is rich in lecithin, and the cell membrane of nerve cells and the membrane on the surface of nerve fibers in the brain contain lecithin. Egg yolk is covered on the surface of primary neuronal cells fixed with fixative. According to the principle of like dissolves like, the lecithin in egg yolk fuses with the primary neuronal cells. Then, Golgi staining solution is added to react. Golgi staining solution stains the primary neuronal cells with the assistance of egg yolk lecithin.

[0017] It should be noted that, to date, no existing technology has disclosed the use of egg yolk for Golgi staining of primary neurons. Current methods for labeling or staining primary neurons mainly involve transfection or specific antibody labeling, and while these methods have achieved good imaging results under fluorescence microscopy, their limitations—slow imaging speed, small field of view, and photobleaching of fluorescence signals during imaging—make them problematic for studying the complete structure of neurons. Golgi staining provides excellent staining results for neuronal structures in tissues, and its results can be used to study neuronal structure. It is also a classic method for statistically analyzing neuronal dendritic spines in scientific research. However, this method has not yet yielded satisfactory results when staining primary neurons.

[0018] This invention innovatively adds egg yolk before Golgi staining to treat primary neurons, improving the staining results of existing Golgi staining methods on primary neurons. It successfully achieves clear observation of complete neuronal structures under a microscope. Experimental verification shows that the Golgi staining method provided by this invention has specificity only for primary neurons, achieving good staining results only on primary neurons and not on ordinary cells. It should be understood that the Golgi staining solution formula is the same as existing technologies. The improvement of this invention mainly focuses on adding egg yolk to treat primary neurons before Golgi staining. Utilizing the principle of "like dissolves like," the lecithin in the egg yolk fuses with the primary neurons, and then the Golgi staining solution is added for the reaction. The Golgi staining solution, assisted by the egg yolk lecithin, stains the primary neurons. Furthermore, this invention further optimizes the method, achieving good staining results for primary neurons.

[0019] Compared with the prior art, the positive and progressive effects of the present invention are as follows:

[0020] 1) By adding egg yolk before Golgi staining, the staining results obtained by the Golgi staining method in primary neuronal cell staining can be improved, and the complete neuronal structure can be clearly observed by optical microscopy imaging;

[0021] 2) The application of advanced synchrotron X-ray imaging technology enables deep penetration, rapid imaging, and a wide field of view. It possesses multi-resolution imaging capabilities, ranging from low to high resolution, allowing for the rapid imaging of high-resolution, complete three-dimensional structures of primary neurons. According to the method provided by this invention, using synchrotron X-ray imaging technology to perform complete three-dimensional structural imaging of primary neurons helps to further analyze the complete structure of neurons and understand their function, filling a significant gap in the current field of neuroscience.

[0022] In summary, the Golgi staining method for primary neurons provided by this invention successfully stains primary neurons, and neuronal dendrites and morphology can be observed under synchrotron X-ray imaging. This lays a solid foundation for further research using synchrotron X-ray to study the complete three-dimensional structure and fine three-dimensional structure of neurons, which will promote human understanding of information communication in the nervous system. This Golgi staining method for primary neurons provided by this invention is expected to play an important role in the field of neuroscience. Attached Figure Description

[0023] Figure 1AThis is an image of primary neurons stained with Golgi apparatus without the addition of egg yolk. Figure 1B This is an image of primary neurons stained with egg yolk and then subjected to Golgi staining.

[0024] Figure 2A , Figure 2B , Figure 2C , Figure 2D These are images of primary neurons stained with egg yolk for different durations of Golgi staining, namely 2h, 4h, 6h, and 24h.

[0025] Figure 3A This is an image of HeLa cells stained with egg yolk and then subjected to Golgi staining. Figure 3B This is an image of PC12 cells stained with Golgi apparatus after adding egg yolk. Figure 3C This is an image of HeLa cells stained with egg yolk and then subjected to Golgi staining.

[0026] Figure 4A , Figure 4B This is a synchrotron X-ray image of primary neurons stained with egg yolk and then subjected to Golgi staining. Figure 4A This is an image of the complete primary neuronal cell structure. Figure 4B This is an image of the dendritic spine structure on a primary neuron. Detailed Implementation

[0027] The present invention will be further illustrated below with specific implementation examples. It should be understood that the following embodiments are for illustrative purposes only and not for limiting the scope of the invention. The following embodiments are experimental methods without specific conditions, following conventional methods and conditions, or selected according to the product instructions. All reagents and raw materials used in this invention are commercially available.

[0028] This invention mainly selects egg yolk as the lecithin that plays a role in experiments and proposes a Golgi staining method for synchrotron radiation X-ray imaging of primary neuronal cells. This method can be used for synchrotron radiation X-ray imaging of the structure of primary neuronal cells. The following examples illustrate the implementation effect of this invention.

[0029] Example 1: Staining primary neurons with egg yolk-assisted Golgi staining solution.

[0030] Pregnant SD mice were purchased from Shanghai Silex Laboratory Animal Co., Ltd. SD mice (P0) were surgically obtained on day 18 of pregnancy. Brain tissue was then surgically extracted from the P0 mice. The brain tissue was placed in centrifuge tubes containing HBSS buffer, and then digested with trypsin and DNase to obtain a tissue suspension. The digestion was then stopped using a culture medium containing MEM Eagle's with Earle's BSS, glucose, fetal bovine serum albumin, antibiotics, and glutamine. At this point, cells had been extracted from the tubes, and their cell density was diluted to 1–3 × 10⁻⁶ cells / mL. 5 / ml. Inoculate into culture dishes containing Neurobasal Mediu, B-27 supplement, glutamine, and antibiotics, and culture for at least 10 minutes before use for subsequent staining experiments. Fix primary neurons with 4% paraformaldehyde at 26°C for 30 min, then remove the paraformaldehyde, quickly rinse with 1xPBS buffer, evenly coat the surface of the primary neurons with egg yolk, incubate at 26°C for 15 min, remove the surface egg yolk, add prepared Golgi staining solution to cover the cell surface, react at 26°C for 24 h, then reduce and develop with ammonia solution at 26°C for 10 min, and finally observe using an inverted optical microscope. For the blank control group, follow the same steps as above, except for the addition of egg yolk.

[0031] The results are as follows Figure 1A , 1B As shown, Figure 1A The results were obtained from Golgi staining of primary neurons without the addition of egg yolk; no neuronal structure was found under the microscope. Figure 1B This is the result of adding egg yolk to primary neurons for Golgi staining. Under a microscope, the neuronal structure can be clearly observed to be stained, and the dendritic structure (red arrow) is clearly visible.

[0032] Example 2 investigates the staining effects of different Golgi staining solutions with varying treatment durations under the influence of egg yolk.

[0033] Cell extraction and culture were the same as in Example 1. When the cells were ready for experimentation, primary neurons were fixed at 26°C for 30 min with 4% paraformaldehyde. After that, the paraformaldehyde was removed, and the cells were quickly rinsed with 1xPBS buffer. Egg yolk was then evenly applied to the surface of the primary neurons. The cells were placed at 26°C for 15 min, and the surface egg yolk was removed. Then, the prepared Golgi staining solution was added to cover the cell surface for reaction. The reaction was carried out at 26°C for 2 h, 4 h, 6 h, and 24 h, respectively. After that, the cells were reduced and developed with ammonia solution at 26°C for 10 min. Finally, the cells were observed using an inverted optical microscope.

[0034] Experimental results are as follows Figure 2A-2D As shown, Figure 2A The image shown is an optical microscope image obtained after staining with Golgi staining solution for 2 hours. No neuronal structures were observed in the image. Figure 2B The image shown is an optical microscope image obtained after staining with Golgi staining solution for 4 hours. Significant neuronal and dendritic structures are visible (red arrows), but some neuronal structures show poor staining, which will affect subsequent data processing and analysis. Figure 2C This image shows the results of optical microscopy imaging after 6 hours of staining with Golgi staining solution. Significant neuronal and dendritic structures (red arrows) are visible in the image, including some longer dendritic structures. The background is also cleaner than the results after 4 hours of staining. Figure 2D The image shown is an optical microscope image of primary neurons stained with Golgi stain for 24 hours. Significant neuronal and dendritic structures (red arrows) are clearly visible, as are the neural networks formed by intertwined nerve fibers. This demonstrates that in subsequent experiments, treating primary neurons with Golgi stain for at least 6 hours will yield satisfactory staining results.

[0035] Example 3 explores the specific staining effect of the optimized Golgi staining method on primary neurons.

[0036] The extraction, culture, and staining process of primary neuronal cells were consistent with the experimental group steps in Example 1. HeLa cells and PC12 cells were preserved in our laboratory, and their related Golgi staining steps were consistent with the Golgi staining steps of primary neuronal cells.

[0037] Experimental results are as follows Figures 3A-3C As shown, Figure 3A The image shown is an optical microscope image obtained by staining HeLa cells using an optimized Golgi staining method. This method did not effectively stain the morphology and structure of HeLa cells, only producing black precipitates in the image. Figure 3B The image shown is an optical microscope image obtained by staining PC12 cells using an optimized Golgi staining method. This method did not effectively stain the morphology and structure of PC12 cells, and only produced black precipitates in the image. Figure 3C The image shown is an optical microscope image obtained by staining primary neurons using the optimized Golgi staining method. The image reveals the complete neuronal structure and dendritic structure. These results demonstrate that the optimized Golgi staining method only stains primary neurons.

[0038] Example 4: Observation of staining results using synchrotron X-ray imaging technology

[0039] Pregnant SD mice were purchased from Shanghai Silex Laboratory Animal Co., Ltd. SD mice (P0) were surgically obtained on day 18 of pregnancy. Brain tissue was then surgically extracted from the P0 mice. The brain tissue was placed in centrifuge tubes containing HBSS buffer, and then digested with trypsin and DNase to obtain a tissue suspension. The digestion was then stopped using a culture medium containing MEM Eagle's with Earle's BSS, glucose, fetal bovine serum albumin, antibiotics, and glutamine. At this point, cells had been extracted from the tubes, and their cell density was diluted to 1–3 × 10⁻⁶ cells / mL. 5 / ml. The cells were seeded into culture dishes containing Neurobasal Mediu, B-27 supplement, glutamine, and antibiotics. These dishes were pre-positioned with a silica window base and cultured in vitro for at least 10 minutes before subsequent staining experiments. Primary neurons were fixed at 26°C with 4% paraformaldehyde for 30 min. After removing the paraformaldehyde, the cells were quickly rinsed with 1xPBS buffer. Egg yolk was then evenly applied to the surface of the primary neurons. The cells were incubated at 26°C for 15 min, after which the surface yolk was removed. Prepared Golgi staining solution was added to cover the cell surface for reaction. The reaction was carried out at 26°C for 12 h. Afterward, reduction and development were performed using an ammonia solution at 26°C for 10 min. Finally, synchrotron X-ray imaging was used for observation.

[0040] Experimental results are as follows Figures 4A-4B As shown, Figure 4A The synchrotron X-ray imaging results were obtained by staining primary neurons using an optimized Golgi staining method. This allows for observation of complete neuronal and dendritic structures with minimal background noise. Further magnification allows for imaging and observation of dendritic spines on the dendrites. Figure 4B This is a structure not observed under an optical microscope. These results demonstrate that the method is applicable to synchrotron radiation X-ray imaging and can perform high-resolution imaging analysis of stained primary neuronal structures.

[0041] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of the invention. Various variations can be made to the above embodiments of the present invention. All simple and equivalent changes and modifications made in accordance with the claims and description of this application fall within the protection scope of the claims of this patent. All aspects not described in detail in this invention are conventional technical content.

Claims

1. A Golgi staining method for primary neurons, characterized in that, Includes the following steps: 1) Extraction and culture of primary neurons from animal brain tissue; 2) The primary neuronal cells were fixed using a fixative solution; 3) The egg yolk is evenly covered on the surface of the primary neuron cells. After reacting for a period of time, the egg yolk is removed, and Golgi staining solution is added to cover the surface of the primary neuron cells for reaction, thereby staining the fixed primary neuron cells. 4) Remove the Golgi stain and react with a reducing agent; and 5) Optical microscopy imaging and / or synchrotron X-ray imaging.

2. The Golgi staining method according to claim 1, characterized in that, In step 3), the egg yolk is reacted for 5 to 60 minutes at a temperature of 10 to 30 degrees Celsius.

3. The Golgi staining method according to claim 1, characterized in that, In step 3), the reaction time of the Golgi staining solution is 0.5h to 72h, and the reaction temperature is 4℃ to 80℃.

4. The Golgi staining method according to claim 1, characterized in that, In step 2), the fixative is selected from: 1% to 12.5% ​​glutaraldehyde, 2% to 10% paraformaldehyde, 5% to 10% formalin or 10% to 100% ethanol.

5. The Golgi staining method according to claim 1, characterized in that, In step 2), the fixation time of the fixative is 10 to 60 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% ammonia aqueous solution or 1% to 10% lithium hydroxide aqueous 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 280 eV to 20 keV.

8. The Golgi staining method according to claim 1, characterized in that, In step 5), the resolution of the synchrotron radiation X-ray imaging is 10nm to 1000nm.

9. The Golgi staining method according to claim 1, characterized in that, In step 1), the animal brain tissue includes: SD mouse brain tissue, C57BL / 6 mouse brain tissue, or BALB / c mouse brain tissue.

10. An application of the Golgi staining method according to any one of claims 1 to 9 in the field of neuroscience, wherein the Golgi staining method can achieve rapid, high-resolution, and complete three-dimensional imaging of primary neuronal cells.

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