Sodium cholate golgi staining solution and staining method for brain tissue staining

Abstract

The application discloses a sodium cholate Golgi staining solution and a staining method for brain tissue staining. The sodium cholate Golgi staining solution is prepared by compounding a basic Golgi-cox staining solution and sodium cholate. The basic Golgi-cox staining solution is prepared by mixing 5% mercuric chloride solution, 5% potassium dichromate solution, 5% potassium chromate solution and deionized water in a volume ratio of 5:5:4:10, and then storing the mixture in dark for at least two days to obtain supernatant. The final concentration of the sodium cholate is 0.25-1 (w / v) %. The application introduces exogenous sodium cholate into the Golgi-cox staining solution for the first time, improves the classical Golgi-cox staining method, shortens the traditional staining period of several weeks to several days, lays a good foundation for studying the action mechanism of the Golgi-cox staining, and provides a simple, low-cost and stable staining technology support for various brain science neuron morphology research.

Description

Technical Field

[0001] This invention relates to the field of bioimaging staining, and more specifically to a sodium cholate Golgi staining solution and staining method for staining brain tissue. Background Technology

[0002] Since its inception in the late 19th century, Golgi staining has been considered the gold standard for neuromorphological research due to its unique advantages in randomly and sparsely labeling neurons and fully displaying the fine structures of neuronal cell bodies, axons, dendrites, and dendritic spines. The subsequently improved Golgi-Cox method, which uses mercuric chloride instead of traditional silver salts for staining, further improved the signal-to-noise ratio and staining success rate, and continues to play an irreplaceable role in basic neuroscience research and clinical pathological analysis. However, Golgi-Cox staining requires an immersion process lasting several weeks, typically 2 to 8 weeks, and in some cases even longer, to allow heavy metal ions to fully penetrate and form sufficient initial binding with neuronal membrane components. Such a lengthy experimental period significantly slows down research progress and can easily lead to problems such as excessive sclerosis of brain tissue, increased fragility, and increased background precipitation, severely limiting its widespread application in modern neuroscience research.

[0003] To address the aforementioned technical bottlenecks, researchers have continuously explored and improved the process. Regarding staining solution formulation, researchers have experimented with adjusting the concentration of the infiltration solution, regulating the pH value, and using different fixatives to immobilize brain tissue, aiming to improve staining results. In terms of physical treatment, measures such as microwave assistance, temperature control, and high pressure have been introduced to accelerate the infiltration process. For example, increasing the infiltration temperature to 37°C accelerates staining. Regarding high pressure assistance, recent studies have shown that under 9 MPa high pressure, the staining time for whole-brain neurons in mice can be reduced to one-quarter of that using traditional methods. Furthermore, some researchers have explored strategies such as embedding brain tissue blocks in egg yolk to optimize staining results.

[0004] However, each of the above-mentioned improvement schemes has significant shortcomings. Simply adjusting the concentration and pH of the impregnation solution or using physical barrier methods such as egg yolk embedding has very limited effect on improving impregnation efficiency and cannot fundamentally solve the problem of excessively long staining time. Fixatives lead to a large number of glial cells being stained, making it difficult to analyze neurons. While high-temperature treatment can shorten the staining cycle, the high-temperature environment can easily cause thermal damage to the fine structures of brain tissue, affecting the integrity of neuronal morphology, and accelerating neuronal staining while also exacerbating vascular staining. Although the high-pressure method is highly effective, it requires specialized high-pressure equipment, making it difficult to implement and widely adopt in routine laboratories. Microwave treatment also requires specialized equipment and has a certain impact on tissue morphology.

[0005] Therefore, it is necessary to develop a simple, economical, more applicable, more stable, non-damaging to tissue structure, and capable of significantly shortening the staining cycle Golgi-cox optimization scheme. Summary of the Invention

[0006] The purpose of this invention is to provide a sodium cholate Golgi staining solution and staining method for brain tissue staining, thereby solving the problems of traditional Golgi-Cox staining with a long immersion period of several weeks, which is time-consuming and easily causes brain tissue sclerosis, increased background precipitation, and cell necrosis. Existing improved methods either damage the fine structure of nerves, require specialized and expensive equipment that is difficult to popularize, or have weak staining effect.

[0007] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0008] According to a first aspect of the present invention, a sodium cholate Golgi staining solution for staining brain tissue is provided, which is prepared by compounding a basic Golgi-cox staining solution with sodium cholate; the basic Golgi-cox staining solution is prepared by mixing 5% mercuric chloride (HgCl2) solution, 5% potassium dichromate (K2Cr2O7) solution, 5% potassium chromate (K2CrO4) solution and deionized water in a volume ratio of 5:5:4:10, and then allowing the mixture to stand and settle in the dark for at least two days before taking the supernatant; the final concentration of sodium cholate in the sodium cholate Golgi staining solution is 0.25~1 (w / v, g / 100 mL)%.

[0009] Most preferably, the final concentration of sodium cholate in the sodium cholate Golgi staining solution is 0.5% (w / v). According to research based on the present invention, excessively high sodium cholate concentrations can excessively damage neuronal membrane structures and inhibit the specific staining of neurons by metal deposits. Furthermore, mixing sodium cholate too early can easily lead to oxidation or non-specific reduction side reactions, resulting in decreased surface activity and ultimately unsuccessful staining.

[0010] Preferably, the sodium cholate Golgi staining solution is prepared and used immediately, and the time from preparation to brain tissue immersion does not exceed 6 hours.

[0011] According to a second aspect of the present invention, a method for staining brain tissue based on the sodium cholate Golgi staining solution is provided, comprising the following steps:

[0012] 1) Prepare and allow the basic Golgi-cox staining solution to stand, and obtain a clear supernatant;

[0013] 2) Add sodium cholate to the supernatant and mix well. Prepare fresh sodium cholate Golgi staining solution.

[0014] 3) Obtain fresh animal brain tissue that has undergone perfusion hemostasis treatment;

[0015] 4) The brain tissue was stained by immersion in the fresh sodium cholate Golgi staining solution prepared in step 2) in the dark at a constant temperature.

[0016] 5) After staining, the brain tissue was dehydrated with sucrose and then prepared into sections using a sectioning device;

[0017] 6) The brain tissue sections are sequentially subjected to imaging, graded alcohol dehydration, clearing, and then mounting or embedding; and

[0018] 7) Perform imaging observation on the processed brain tissue slices.

[0019] The novel sodium cholate Golgi staining method (SC Golgi-cox) for brain tissue staining provided by the present invention works as follows: Sodium cholate (SC) is an amphiphilic molecule belonging to the bile acid salt class. While enhancing the permeability of cell membranes and extracellular matrix to water-soluble heavy metal ions, it guides ions to be directionally enriched along the neuronal membrane system, so that the freshly prepared modified SC-Golgi staining solution can significantly accelerate the effective immersion speed.

[0020] Preferably, in step 3), the fresh animal brain tissue sample includes complete brain tissue or brain tissue slices selected from: fruit flies, zebrafish, mice, rats, rabbits, cats, dogs, pigs, monkeys, or humans.

[0021] Preferably, in step 4), the immersion and staining conditions are constant temperature incubation at 26 ℃ in the dark for 5 to 10 days.

[0022] Preferably, in step 4), modified SC-Golgi staining solution is used to immerse and stain brain tissue. The staining time varies for different animal brain tissues; the optimal staining time for mouse brain tissue is 7 days. Experiments of this invention have demonstrated that staining mouse brain tissue with modified SC-Golgi staining solution for 7 days resulted in better neuronal morphological structure integrity compared to staining with traditional Golgi-cox staining solution for 7 days, and was comparable to staining with traditional Golgi-cox staining solution for 14 days.

[0023] Preferably, in step 5), the animal tissue should be soaked and washed with ultrapure water or 30% sucrose solution before sectioning to remove excess Golgi staining solution.

[0024] Preferably, in step 5), the slicing equipment is selected from cryostats, vibratory slicing machines, ultrathin slicing machines, or paraffin slicers, and the slice thickness ranges from 5 nm to 5000 μm.

[0025] For optical microscopy, the optimal slice thickness is 100 μm, while for X-ray microscopy, 1 mm to 2 mm thick slices or whole brain tissue are often selected.

[0026] Preferably, in step 6), the developing solution used is a 15%~18% ammonia solution and a 5% sodium thiosulfate solution; the frozen sections are first treated with ammonia for 8~12 min, washed with water, and then treated with sodium thiosulfate for 8~12 min and washed with water to complete the neuronal development.

[0027] According to a preferred embodiment of the present invention, for 100 μm frozen sections, the treatment is ammonia water for 10 min, ultrapure water for 5 min, sodium thiosulfate for 10 min, and ultrapure water for 5 min.

[0028] Preferably, in step 6), the gradient dehydration alcohol is 30%, 50%, 75%, 90%, and 100% ethanol, and the dehydration time for each concentration of alcohol is 10 min to 24 h.

[0029] Preferably, in step 6), the mounting is performed by soaking the sample in xylene for 15 min and then using a neutral resin or a fast-sealing desiccant for optical imaging.

[0030] Preferably, in step 6), the embedding is performed using paraffin or epoxy resin for synchrotron X-ray imaging samples.

[0031] Preferably, in step 6), for paraffin-embedded tissue, after gradient dehydration, it is permeated with xylene for 2-6 h, then soaked in paraffin for 4-12 h, and then cured at room temperature for 24 h for embedding.

[0032] Preferably, in step 6), for epoxy resin embedded tissue, after gradient dehydration, it is soaked in acetone for 12 hours, then soaked in acetone:resin gradient, and finally soaked in resin for 24 hours, and then cured at 70°C for 48 hours for embedding.

[0033] Preferably, in step 7), microscopic imaging observation is divided into two categories: optical microscopy imaging and synchrotron X-ray imaging. Optical microscopy imaging refers to microscopic imaging based on optical principles. Common equipment includes ordinary optical microscopes (such as Zeiss optical microscopes), laser scanning confocal microscopes, light sheet microscopes, and super-resolution optical microscopes. Synchrotron X-ray imaging is an imaging platform that relies on synchrotron accelerator devices to carry out X-ray imaging. Common methods include X-ray absorption imaging, X-ray phase-contrast imaging, and X-ray fluorescence imaging. These imaging beamlines can perform two-dimensional or three-dimensional X-ray imaging of brain tissue, and can acquire brain neural connectivity imaging data that meets the resolution requirements, thereby obtaining the corresponding brain connectivity atlas.

[0034] Sodium cholate is an amphiphilic molecule belonging to the bile acid salt class, possessing both excellent biocompatibility and interfacial activity: its hydrophobic end can gently embed into the lipid bilayer of the cell membrane, resulting in a loose lipid arrangement, significantly improving cell membrane permeability without damaging the overall neuronal structure. In existing technologies, sodium cholate has been used in OptiMuS-prime tissue clearing technology to achieve lipid removal, membrane protein dissolution, and purification. This invention adds sodium cholate to the supernatant of Golgi-cox staining solution as a staining promoter. Without damaging the structural integrity of neurons, it accelerates the penetration of heavy metal ions by increasing cell membrane permeability, promoting rapid binding of heavy metals to neurons and completing staining. This chemical mechanism significantly improves the impregnation efficiency, fundamentally solving the technical problem of the time-consuming traditional Golgi staining process.

[0035] The research presented in this invention confirms that the modified SC-Golgi staining solution can significantly shorten the staining cycle while effectively maintaining the stability of the neuronal structure in the tissue. This modified SC-Golgi staining solution is suitable for rapid staining of various animal brain tissue samples; especially for large-volume brain tissue, short-time immersion avoids problems such as cell necrosis and staining failure caused by long-term staining using traditional methods, demonstrating promising prospects for biological applications.

[0036] The key invention of this invention lies in the use of sodium cholate to improve the traditional Golgi-cox staining system. Sodium cholate can not only change the permeability of neuronal membranes, but also promote the directional enrichment of heavy metal ions. While accelerating the staining speed, it also provides a new research idea for elucidating the intrinsic mechanism of Golgi-cox-specific labeling of neurons.

[0037] Compared with the prior art, the positive and progressive effects of the present invention are as follows:

[0038] 1) Significantly reduced staining cycle: Traditional Golgi-Cox staining requires waiting for heavy metal ions to slowly penetrate and establish equilibrium with the neuronal membrane, which takes about 2 to 8 weeks. The long time cost severely limits the speed of experimental iteration and the conduct of large-scale studies. The addition of sodium cholate in this invention can gently improve membrane permeability, and staining can be completed in just a few days. While ensuring staining quality, it supports rapid phenotypic screening and large-scale comparison of neuronal morphology before and after drug administration, effectively improving the experimental throughput and efficiency of neuromorphological studies.

[0039] 2) Complete preservation of fine neural structures: Although existing high-pressure assisted modification schemes can shorten staining time, the pressurization and depressurization operations can easily cause damage and deformation of brain tissue; This invention relies on the gentle action of sodium cholate small molecules on the lipid bilayer, which can accelerate the orderly deposition of metal ions along the neuronal membrane at room temperature or slightly below room temperature, completely preserving the fine morphology of dendritic spines, and is suitable for uniform and rapid staining of ultra-thick tissue blocks, thus broadening the applicable scenarios of Golgi staining.

[0040] 3) Providing a new model for mechanism research: Traditional Golgi-Cox staining is a multi-component, heterogeneous precipitation system in which multiple factors such as heavy metal ion penetration, local supersaturation, nucleation selectivity, and colloidal stability interact, and its specific staining mechanism is still unclear; sodium cholate directly changes the staining effect by regulating cell membrane permeability, and can be used as a tool to explore the molecular mechanism of Golgi staining.

[0041] In summary, this invention provides a sodium cholate Golgi staining solution and a staining method for staining brain tissue. This invention, for the first time, incorporates sodium cholate, an amphiphilic bile salt molecule with both interfacial activity and good biocompatibility, as a staining promoter. This is added to the supernatant of traditional Golgi-cox staining solution after static settling, resulting in a modified sodium cholate SC-Golgi staining solution. Utilizing the hydrophobic end of sodium cholate's ability to gently insert into the lipid bilayer of neuronal cell membranes and enhance membrane permeability, it accelerates the directional accumulation of heavy metal ions into the neuronal membrane, thus establishing a novel Golgi staining method for brain tissue. This method can shorten the traditional staining cycle of several weeks (e.g., 2 weeks) to several days (e.g., 7 days), enabling rapid staining of various animal brain tissue samples under room temperature conditions. It avoids the problems of tissue cell necrosis and staining failure caused by long-term immersion, and does not cause brain tissue damage like other modified methods such as high pressure. It can completely preserve the fine structures of neurons, such as dendritic spines, and is compatible with various detection methods such as frozen sections, light microscopy of thick tissue blocks, and synchrotron X-ray imaging. This not only significantly increases the experimental throughput of neuromorphological research but also alters the staining effect by regulating cell membrane permeability, providing insights into the analysis of Golgi-cox... The staining-specific labeling of neurons provides a novel research vehicle for understanding the intrinsic mechanisms of neuronal morphology, and can provide simple, low-cost, and highly stable staining techniques to support various neuronal morphology studies in brain science. Attached Figure Description

[0042] Figure 1 The results of staining mouse brain tissue with modified SC-Golgi staining solution and conventional Golgi-cox staining solution are shown. A is an optical microscopic image of the cerebral cortex region of a mouse brain stained with fresh modified SC-Golgi (0.5% SC) staining solution for 7 days; B is an optical microscopic image of the cerebral cortex region of a mouse brain stained with conventional Golgi-cox staining solution for 7 days; and C is an optical microscopic image of the cerebral cortex region of a mouse brain stained with conventional Golgi-cox staining solution for 14 days.

[0043] Figure 2Light microscopy results of mouse brain tissue stained with modified SC-Golgi staining solution at different concentrations of sodium cholate; where AF shows light microscopic images of the cerebral cortex region of fresh modified SC-Golgi stained mouse brain tissue with sodium cholate concentrations of 0, 0.25%, 0.5%, 0.75%, 1%, and 2%, respectively.

[0044] Figure 3 Light microscopy results of mouse brain tissue stained with conventional Golgi-cox staining solution, fresh modified SC-Golgi staining solution, and modified SC-Golgi staining solution premixed with sodium cholate. A shows an optical microscopic image of the cerebral cortex region of a mouse brain stained with conventional Golgi-cox staining solution 7 days later; B shows an optical microscopic image of the cerebral cortex region of a mouse brain stained with fresh modified SC-Golgi (0.5% SC) staining solution 7 days later; C shows an optical microscopic image of the cerebral cortex region of a mouse brain stained with modified SC-Golgi (0.5% SC) staining solution premixed with sodium cholate 7 days later.

[0045] Figure 4 Two-dimensional synchrotron X-ray imaging results of mouse brain tissue stained with modified SC-Golgi (1% SC) staining solution. Detailed Implementation

[0046] The present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the techniques used in the embodiments are conventional practices in the art, or experimental methods recommended by the reagent kit and instrument manufacturers. Unless otherwise specified, the reagents and materials used in the embodiments are commercially available.

[0047] Example 1

[0048] This example is used to compare the effects of staining mouse brain tissue with fresh modified SC-Golgi staining solution for 7 days, staining mouse brain tissue with conventional Golgi-cox staining solution for 7 days, and staining mouse brain tissue with conventional Golgi-cox staining solution for 14 days.

[0049] Prepare traditional Golgi-cox staining solution and fresh modified SC-Golgi staining solution respectively.

[0050] The traditional method for preparing Golgi-cox staining solution is as follows: First, prepare three aqueous solutions of 5% potassium dichromate, 5% mercuric chloride, and 5% potassium chromate respectively. Then, mix the three stock solutions with deionized water in a volume ratio of 5:5:4:10. After mixing, transfer the solution to a brown bottle and let it stand at room temperature in the dark for at least 48 hours until the heavy metal deposits have settled. Only the clear supernatant is used, which is the traditional Golgi-cox staining solution required for the experiment.

[0051] The preparation method of fresh modified SC-Golgi (0.5% SC) staining solution is as follows: Take a 20 mL brown vial (for staining mouse brain tissue), weigh 0.035 g sodium cholate into the brown vial, then measure 7 mL of the supernatant of the traditional Golgi-cox staining solution that has been prepared in advance and allowed to stand for at least 48 h, mix well and the fresh modified SC-Golgi (0.5% SC) staining solution is prepared for staining.

[0052] Mice were deeply anesthetized with 1% pentobarbital. Following standard experimental procedures, blood was removed by cardiac perfusion with saline solution. Mouse brain tissue was quickly extracted and placed in fresh, modified SC-Golgi (0.5% SC) staining solution. The tissue was then incubated at 26°C for 7 days in the dark. After staining, the brain tissue blocks were removed and dehydrated in a 30% sucrose solution at 4°C in the dark for approximately 3 days, with the 30% sucrose solution replaced every 24 hours. In this embodiment, frozen sections were prepared, with coronal sections 100 μm thick. The prepared mouse brain tissue sections were reacted with 18% ammonia for 10 minutes, washed with water, and then reacted with 5% sodium thiosulfate for 10 minutes. The reaction was terminated after washing with water. The slides were then dehydrated using a series of gradient alcohols of 50%, 75%, 90%, and 100%, with each gradient dehydration time being 5 minutes. After that, xylene was used for clearing, which took at least 15 minutes. Finally, the slides were mounted with a rapid mounting desiccant and observed using a Nikon optical microscope after the xylene had completely evaporated. Figure 1 Image A shows an optical microscopic image of the cerebral cortex region of a mouse brain stained with fresh modified SC-Golgi staining solution for 7 days.

[0053] Mice were deeply anesthetized with 1% pentobarbital. Following standard experimental procedures, blood was removed by cardiac perfusion with saline. Brain tissue was then rapidly extracted and placed in conventional Golgi-Cox staining solution, and stained in a 26°C incubator for 7 and 14 days, protected from light. Subsequent procedures were the same as described above. Figure 1 Image B shows an optical microscopic image of the cerebral cortex region of a mouse brain stained with conventional Golgi-cox staining solution for 7 days. Figure 1Image C shows an optical microscopic image of the cerebral cortex region of a mouse brain stained with conventional Golgi-cox staining solution for 14 days.

[0054] As shown in the figure, brain tissue stained with fresh modified SC-Golgi staining solution for 7 days showed significantly better neuronal morphological and structural integrity than neurons stained with traditional Golgi-cox staining solution for 7 days, and the effect was comparable to neurons stained with traditional Golgi-cox staining solution for 14 days.

[0055] Example 2

[0056] This example explores the effect of modified SC-Golgi staining solution with different sodium cholate concentrations on the staining effect on mouse brain tissue.

[0057] Fresh modified SC-Golgi staining solutions with different concentrations of sodium cholate were prepared.

[0058] The preparation method of fresh modified SC-Golgi staining solutions with different sodium cholate concentrations is the same as in Example 1, except that the only difference is the mass of sodium cholate. Weigh 0 g, 0.0175 g, 0.035 g, 0.0525 g, 0.07 g, and 0.14 g of sodium cholate, respectively, and then add them to 7 mL of the supernatant of traditional Golgi-cox staining solution that has been prepared in advance and allowed to stand for at least 48 h. After mixing, fresh modified SC-Golgi staining solutions with sodium cholate concentrations of 0%, 0.25%, 0.5%, 0.75%, 1%, and 2% can be prepared.

[0059] Mice were deeply anesthetized with 1% pentobarbital. Following standard experimental procedures, blood was removed by cardiac perfusion with saline solution. Mouse brain tissue was quickly extracted and placed in prepared modified SC-Golgi staining solution with different concentrations of fresh sodium cholate (0, 0.25%, 0.5%, 0.75%, 1%, 2%). The tissue was then incubated at 26°C for 7 days in the dark. After staining, the brain tissue blocks were removed and dehydrated in a 30% sucrose solution at 4°C in the dark for approximately 3 days, with the 30% sucrose solution replaced every 24 hours. In this embodiment, frozen sections were prepared, with coronal sections 100 μm thick. The prepared mouse brain tissue sections were reacted with 18% ammonia for 10 minutes, washed with water, and then reacted with 5% sodium thiosulfate for 10 minutes. The reaction was terminated after washing with water. The slides were then dehydrated using a series of gradient alcohols of 50%, 75%, 90%, and 100%, with each gradient dehydration time being 5 minutes. After that, xylene was used for clearing, with a clearing time of at least 15 minutes. Finally, the slides were mounted with a rapid mounting desiccant. After the xylene had completely evaporated, the slides were observed using a Nikon optical microscope. Figure 2The images shown in the middle AF are optical microscopic images of the cerebral cortex region of mouse brains stained with fresh modified SC-Golgi (0, 0.25%, 0.5%, 0.75%, 1%, 2%).

[0060] As shown in the figure, the staining effect of fresh modified SC-Golgi staining solution varies depending on the SC concentration. Fresh modified SC-Golgi staining solution (0.5% SC) produces the most neurons and the most complete dendritic structure, resulting in the best optimization effect. Concentrations of too low a concentration (0, 0.25% SC) result in insignificant optimization, fewer neurons, shorter dendritic fibers, and incomplete neuronal structure. Concentrations of too high a concentration (1%, 2% SC) excessively damage neuronal structure and produce irregular precipitates, leading to poor staining results. According to this embodiment, the SC concentration in fresh modified SC-Golgi staining solution is preferably 0.5%~0.75%, and most preferably 0.5%.

[0061] Example 3

[0062] This example compares the staining effects of traditional Golgi-cox staining solution, fresh modified SC-Golgi staining solution, and modified SC-Golgi staining solution premixed with sodium cholate on mouse brain tissue.

[0063] Take a 20 mL brown vial and measure 7 mL of the supernatant of traditional Golgi-Cox staining solution, which has been prepared in advance and allowed to stand for at least 48 h, for staining. Mice were deeply anesthetized with 1% pentobarbital. Following experimental procedures, blood was removed by cardiac perfusion with physiological saline. Mouse brain tissue was quickly removed and placed in a modified SC-Golgi staining solution pre-mixed with SC. The tissue was then incubated at 22°C for 7 days in the dark. After staining, the brain tissue blocks were removed and placed in a 30% sucrose solution at 4°C in the dark for approximately 3 days to dehydrate. The 30% sucrose solution was replaced every 24 h. In this example, frozen sections were prepared. The sections were coronal in shape and 100 μm thick. The prepared mouse brain tissue sections were reacted with 18% ammonia for 10 minutes, washed with water, and then reacted with 5% sodium thiosulfate for 10 minutes. The reaction was terminated after washing with water. The slides were then dehydrated using a series of gradient alcohols of 50%, 75%, 90%, and 100%, with each gradient dehydration time being 5 minutes. After that, xylene was used for clearing, which took at least 15 minutes. Finally, the slides were mounted with a rapid mounting desiccant and observed using a Nikon optical microscope after the xylene had completely evaporated. Figure 3 Image A shows an optical microscopic image of the cerebral cortex region of a mouse brain stained with conventional Golgi-cox staining solution for 7 days.

[0064] Take a 20 mL brown vial, weigh 0.035 g of sodium cholate into the vial, and measure 7 mL of the supernatant of the traditional Golgi-cox staining solution that has been prepared in advance and allowed to stand for at least 48 h. Mix well to prepare a fresh modified SC-Golgi (0.5% SC) staining solution for staining. Mice were deeply anesthetized with 1% pentobarbital. According to experimental procedures, blood was removed by perfusion of the heart with physiological saline. The mouse brain tissue was quickly removed and placed in the modified SC-Golgi staining solution mixed with SC. It was then placed in a 22°C incubator for staining for 7 days in the dark. After staining, the brain tissue block was removed and placed in a 30% sucrose solution at 4°C in the dark for dehydration for about 3 days. The 30% sucrose solution was changed every 24 h. In this embodiment, frozen sections were used. The sections were coronal in shape and 100 μm thick. The cut mouse brain tissue sections were reacted with 18% ammonia for 10 minutes, washed with water, and then reacted with 5% sodium thiosulfate for 10 minutes. The reaction was terminated after washing with water. Subsequently, the sections were dehydrated using a series of gradient alcohols (50%, 75%, 90%, and 100%), with each gradient dehydration time being 5 minutes. Afterward, the sections were cleared with xylene for at least 15 minutes. Finally, the sections were mounted with a rapid mounting desiccant. After the xylene had completely evaporated, the sections were observed using a Nikon optical microscope. Figure 3 Image B shows an optical microscopic image of the cerebral cortex region of a mouse brain stained with fresh modified SC-Golgi (0.5% SC) staining solution for 7 days.

[0065] Take a 20 mL brown vial (for mouse brain staining), and add sodium cholate to prepare a modified SC-Golgi (0.5% SC) staining solution premixed with SC. After allowing the metal deposits to settle naturally in the dark for at least 48 hours, dispense the supernatant into the brown vial for staining. Mice were deeply anesthetized with 1% pentobarbital. Following experimental procedures, blood was removed by perfusion of the heart with physiological saline. The mouse brain tissue was quickly removed and placed in the modified SC-Golgi staining solution premixed with SC. It was then stained in a 26°C incubator for 7 days in the dark. After staining, the brain tissue block was removed and placed in a 30% sucrose solution at 4°C in the dark for approximately 3 days to dehydrate. The 30% sucrose solution was replaced every 24 hours. In this embodiment, frozen sections were used. The sections were coronal in shape and 100 μm thick. The cut mouse brain tissue sections were reacted with 18% ammonia for 10 minutes, washed with water, and then reacted with 5% sodium thiosulfate for 10 minutes. The reaction was terminated after washing with water. Subsequently, the sections were dehydrated using a series of gradient alcohols (50%, 75%, 90%, and 100%), with each gradient dehydration time being 5 minutes. Afterward, the sections were cleared with xylene for at least 15 minutes. Finally, the sections were mounted with a rapid mounting desiccant. After the xylene had completely evaporated, the sections were observed using a Nikon optical microscope. Figure 3 Image C shows an optical microscopic image of the cerebral cortex region of a mouse brain stained with a modified SC-Golgi (0.5% SC) staining solution that was premixed with SC seven days after staining.

[0066] As shown in the figure, pre-mixing SC in the modified SC-Golgi staining solution resulted in fewer stained neurons, lower structural integrity, and poorer staining effects. We hypothesize that premature mixing of SC may lead to oxidation or non-specific reduction side reactions, resulting in decreased surface activity and ultimately failing to enhance cell membrane permeability, thus preventing heavy metal ions from successfully impregnating and staining neurons. This example demonstrates that pre-mixed SC in the modified SC-Golgi staining solution produces poor staining results in mouse brain tissue, while fresh modified SC-Golgi staining solution shows better neuronal structural integrity.

[0067] Example 4

[0068] In this embodiment, mouse brain tissue was stained with a modified SC-Golgi staining solution and then subjected to synchrotron radiation X-ray imaging to achieve imaging analysis of neuronal structures in the brain tissue.

[0069] Mice were deeply anesthetized with 1% pentobarbital. Following standard experimental procedures, blood was removed by cardiac perfusion with saline solution. Mouse brain tissue was quickly extracted and placed in fresh, modified SC-Golgi (1% SC) staining solution. The tissue was then incubated at 26°C for 7 days in the dark. After staining, the brain tissue blocks were removed and dehydrated in a 30% sucrose solution at 4°C in the dark for approximately 3 days, with the 30% sucrose solution replaced every 24 hours. In this embodiment, sections were prepared manually, in coronal shape, with a thickness of 1 mm. The sectioned mouse brain tissue was reacted with 18% ammonia for 4 hours, washed with water for 1 hour, reacted with 5% sodium thiosulfate for 1 hour, washed with water for another 1 hour, and then the reaction was terminated. Subsequently, dehydration was performed using a series of gradient alcohols at 30%, 50%, 75%, 90%, and 100%, with each gradient dehydration time being 3 hours. Overnight incubation at a 75% alcohol gradient was possible in between. The sample was then impregnated with acetone overnight, followed by impregnation with acetone:resin in ratios of 3:1, 1:1, and 1:3 for 3 hours each, and finally impregnated with resin for 24 hours. All these processes were performed in a shaker at 26 °C and 40 rpm in the dark. Finally, the sample was cured and embedded at 70 °C for 48 hours. Synchrotron X-ray imaging of the sample was then performed using the 13HB beamline of the Shanghai Synchrotron Radiation Facility at an imaging energy of 15 keV and a 10x imaging lens. Figure 4 The image shown is a two-dimensional synchrotron X-ray imaging result of mouse brains stained with fresh modified SC-Golgi (1% SC) staining solution.

[0070] As shown in the figure, fresh modified SC-Golgi staining solution can be used for synchrotron radiation X-ray brain imaging of mouse brains. High-contrast imaging of brain tissue can be observed under high magnification, and a large number of neurons are stained. The results of synchrotron radiation imaging are consistent with those of optical microscopy.

[0071] 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 sodium cholate Golgi staining solution for staining brain tissue, characterized in that, The staining solution is prepared by combining basic Golgi-cox staining solution with sodium cholate. The basic Golgi-cox staining solution is prepared by mixing 5% mercuric chloride solution, 5% potassium dichromate solution, 5% potassium chromate solution, and deionized water in a volume ratio of 5:5:4:10, allowing the mixture to stand and settle in the dark for at least two days, and then collecting the supernatant. The final concentration of sodium cholate in the sodium cholate Golgi staining solution is 0.25~1 (w / v)%. The sodium cholate Golgi staining solution is prepared and used immediately, and the time from preparation to brain tissue immersion should not exceed 6 hours.

2. The sodium cholate Golgi staining solution according to claim 1, characterized in that, The final concentration of sodium cholate in the sodium cholate Golgi staining solution was 0.5 (w / v)%.

3. A method for staining brain tissue using sodium cholate Golgi staining solution for brain tissue staining as described in any one of claims 1 to 2, characterized in that, Includes the following steps: 1) Prepare and allow the basic Golgi-cox staining solution to stand, and obtain a clear supernatant; 2) Add sodium cholate to the supernatant and mix well. Prepare fresh sodium cholate Golgi staining solution. 3) Obtain fresh animal brain tissue that has undergone perfusion hemostasis treatment; 4) Use the fresh sodium cholate Golgi staining solution prepared in step 2) to stain brain tissue by immersion in the dark and at a constant temperature. The sodium cholate Golgi staining solution should be prepared and used immediately. The time from preparation to immersion in brain tissue should not exceed 6 hours. The immersion staining conditions are constant temperature incubation at 26°C in the dark for 5 to 10 days. 5) After staining, the brain tissue was dehydrated with sucrose and prepared into sections using a sectioning device; 6) The brain tissue sections are sequentially subjected to imaging, graded alcohol dehydration, clearing, and then mounting or embedding; and 7) Conduct imaging observations on the processed brain tissue slices.

4. The brain tissue staining method according to claim 3, characterized in that, In step 3), the fresh animal brain tissue sample includes complete brain tissue selected from: fruit fly, zebrafish, mouse, rat, rabbit, cat, dog, pig, monkey or human, or brain tissue slices selected from: fruit fly, zebrafish, mouse, rat, rabbit, cat, dog, pig, monkey or human.

5. The brain tissue staining method according to claim 3, characterized in that, In step 5), the slicing equipment is selected from cryostats, vibratory slicing machines, ultrathin slicing machines, or paraffin slicers, and the slice thickness ranges from 5 nm to 5000 μm.

6. The brain tissue staining method according to claim 3, characterized in that, In step 6), the developing solution used is a mixture of 15%~18% ammonia and 5% sodium thiosulfate. The frozen sections are first treated with ammonia for 8~12 min, washed with water, and then treated with sodium thiosulfate for 8~12 min and washed with water to complete the neuronal development.

7. The brain tissue staining method according to claim 3, characterized in that, In step 6), the gradient dehydration alcohol is 30%, 50%, 75%, 90%, and 100% ethanol, and the dehydration time for each gradient is 5 min to 24 h.

8. The brain tissue staining method according to claim 3, characterized in that, In step 7), the imaging methods include optical microscopy imaging and synchrotron radiation X-ray imaging; optical microscopy includes ordinary optical microscopy, laser scanning confocal microscopy, light sheet microscopy, and super-resolution microscopy; synchrotron radiation X-ray imaging includes X-ray absorption imaging, X-ray phase-contrast imaging, or X-ray fluorescence imaging, which are used to obtain two-dimensional or three-dimensional neural connectivity maps of brain tissue.

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