CN113281130A - Efficient Golgi staining method for large-size brain tissue - Google Patents
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Abstract
本发明提供一种用于大尺寸脑组织的高效的高尔基染色方法，包括以下步骤：1)提供一种加压设备；2)获取动物脑组织；3)配置高尔基染色液，将所述动物脑组织浸染于所述高尔基染色液中，在所述加压设备中进行加压，加压压力为1MPa‑100MPa，加压浸染时间为1‑30天；4)对所述动物脑组织进行切片；5)对切片的动物脑组织进行染色、脱水和包埋；以及6)光学显微镜成像观察和/或同步辐射X射线成像观察。本发明通过对传统的高尔基染色方法进行优化，提高神经元的标记效率、减少染色时间以及提高神经纤维在X光照射下的对比度，拓展了在脑组织的光学成像以及X射线成像方面的应用。
The present invention provides an efficient Golgi staining method for large-sized brain tissue, comprising the following steps: 1) providing a pressurized device; 2) obtaining animal brain tissue; The tissue is immersed in the Golgi staining solution, and pressurized in the pressurized equipment, the pressurization pressure is 1MPa-100MPa, and the pressurization time is 1-30 days; 4) The animal brain tissue is sectioned; 5) Staining, dehydrating and embedding the sliced animal brain tissue; and 6) Observing by optical microscope imaging and/or synchrotron radiation X-ray imaging. By optimizing the traditional Golgi staining method, the invention improves the labeling efficiency of neurons, reduces the staining time and improves the contrast of nerve fibers under X-ray irradiation, thereby expanding the application in optical imaging of brain tissue and X-ray imaging.
Description
Technical Field
The invention relates to the technical field of biomedicine, in particular to a high-efficiency Golgi staining method for large-size brain tissue.
Background
Despite the twenty-first century, the brain is an unsolved fan for humans. For humans, the brain is composed of billions of neurons interconnected by trillions of synaptic structures into intricate neural circuits. The material basis for the realization of the higher functions of the brain is these neural circuits, and the functional basis is the information transmission and information processing processes performed by these neural circuits. Therefore one of the main objectives of modern neuroscience is: the method comprises the following steps of describing a connection map of the neural circuit, understanding the composition rule of the neural circuit, disclosing the mechanism of information transmission and processing in the neural circuit and exploring how the neural circuit finally realizes consciousness and behavior. Therefore, in brain research, it is a very important and challenging loop to study the functional connections of cranial nerves at the level of circuits and even the whole brain.
The study of the neural connectivity map relies on the rapid development of imaging techniques. At the microscopic scale, electron microscopy is the highest resolution imaging tool to date, and some improved electron microscopy has also been used for the study of neural circuits in recent years. However, the imaging range and flux of the method are small due to the imaging principle of the electron microscope, the imaging range of the method is limited to about 0.1mm, and 1 cubic millimeter electron microscope requires about 5 years of work of 1.5 ten thousand people according to the estimation of scientists. Therefore, the study of the neuro-linkage atlas by using an electron microscope cannot realize the large-scale imaging. The resolution of a common optical microscope is limited by diffraction limit, and the current fastest optical imaging technology such as a lattice layer optical microscope can be applied to mouse whole brain imaging, but the resolution of the common optical microscope in the Z-axis direction is only a few microns, so that the brain neural circuit atlas is difficult to perform finer imaging analysis. The X-ray imaging technology based on the synchrotron radiation is the third road in the field of whole brain imaging, the X-ray imaging has the characteristics of high resolution in the same direction, high penetrability, 3D imaging and the like, tissue slices and transparency are not needed in the X-ray imaging, photobleaching is avoided, and quick and long-time imaging can be realized. Therefore, the synchronous radiation-based X-ray imaging method has obvious advantages and broad prospects in brain tissue imaging.
The key to realizing the X-ray imaging of the brain tissue is to improve the contrast of the brain tissue under the X-ray irradiation. Golgi staining is a traditional method for staining nerve cell bodies and nerve fibers of brain tissues. In 1873, the potassium chromate-silver immersion method was invented by the italian scientist Golgi (Golgi), which for the first time realized sparse labeling of neurons in brain tissue and observed a more complete morphological structure of neuron cell bodies and nerve fibers under an optical microscope. Spanish scientist Cajal utilizes the Golgi staining method to observe and describe the morphology of different types of neurons of brain tissues, and improves and consolidates the neurone theory (the neuron coefficient), namely, a nervous system consists of independent neurons, and the neurons or effector cells are in signal transmission through contact, so that the initial theoretical basis of connected omics is laid. The Golgi staining method is a brain tissue staining method based on heavy metal chromium, silver, mercury or the like, and nerve cells of the brain tissue stained by the heavy metal can form contrast with unstained tissues under the irradiation of X-rays, so that X-ray imaging can be performed. However, the traditional Golgi dyeing method has obvious defects: the efficiency of dyeing and marking is low, only about 5 percent of neurons are dyed in each dyeing, and the dyeing is random and has poor repeatability; the dyeing time is long, and can be as long as half a month or several months. In addition, the staining of nerve fibers is less pronounced than that of neuronal cells, and the contrast of nerve fibers is not apparent under X-rays, so that the finer nerve fibers cannot be X-ray imaged. Therefore, at present, further optimization of the traditional Golgi staining method is urgently needed, and a novel efficient Golgi staining method for large-size brain tissue is established, so that the novel Golgi staining method can be applied to optical microscope imaging, is suitable for synchrotron radiation X-ray brain tissue imaging, and realizes the rapid X-ray imaging of the mesoscale nerve connection of the whole brain tissue.
Disclosure of Invention
The invention aims to provide an efficient Golgi staining method for large-size brain tissue, thereby solving the problem that the traditional Golgi staining method is insufficient in brain tissue imaging.
In order to solve the technical problems, the invention adopts the following technical scheme:
provided is a high-efficiency Golgi staining method for large-size brain tissue, which is characterized by comprising the following steps: 1) providing a pressurizing apparatus; 2) obtaining animal brain tissue; 3) preparing a Golgi staining solution, immersing the animal brain tissue in the Golgi staining solution, and pressurizing in a pressurizing device, wherein the pressurizing pressure is 1-100 MPa, and the pressurizing immersion time is 1-30 days; 4) slicing the animal brain tissue; 5) staining, dehydrating and embedding the sliced animal brain tissue; and 6) optical microscopy imaging and/or synchrotron radiation X-ray imaging.
According to the method provided by the invention, the working principle is as follows: the high pressure provided by the high pressure device can integrally improve the heavy metal impregnation proportion of the neurons, promote the permeation of the Golgi staining solution in the brain tissue neurons, increase the impregnation amount of the heavy metal of the nerve fibers, and improve the contrast of the neuron fibers under the X-ray irradiation, thereby realizing the optical microscope imaging and the X-ray imaging observation of the brain tissue.
The pressurizing device in the step 1) comprises a gas pressurizing device and a liquid pressurizing device. The pressurizing device may be purchased commercially or customized to the company.
At present, the pressurizing operation is mainly used for food autoclaving and fresh-keeping, but the method for tissue impregnation, particularly for Golgi dyeing of brain tissue, has not been reported in documents before. Although the traditional Golgi staining method has been used for more than 100 years so far, the problems of too few neurons (5%), randomness of staining, too long time consumption of staining time and the like exist all the time when the brain tissue is stained, and the application of the method in biology, particularly brain science, is severely limited.
The animal brain tissue in the step 2) comprises: drosophila brain tissue, zebrafish brain tissue, mouse brain tissue, rat brain tissue, rabbit brain tissue, cat brain tissue, dog brain tissue, monkey brain tissue, and the like.
The method for obtaining the brain tissue can adopt a buffer solution or a fixed solution heart perfusion method, the brain tissue is taken out quickly according to the conventional experiment, and the brain tissue is carefully operated to avoid being damaged. The animal brain tissue in the step 2) comprises fresh unfixed animal brain tissue or animal brain tissue fixed by adopting a fixing solution, wherein the fixing solution comprises: paraformaldehyde, glutaraldehyde, ethanol, methanol, glacial acetic acid, acetone, formalin, and the like. Among these, paraformaldehyde is most preferred, particularly 4% paraformaldehyde fixing fluid. The paraformaldehyde fixing liquid fixes the protein in the brain tissue, maintains the structure of the brain tissue, and is favorable for further dehydration and observation.
The composition of the Golgi staining solution in the step 3) is as follows: 1) 5% of potassium dichromate, 5% of mercury chloride and 5% of potassium chromate; or 2) 5% potassium dichromate, 1% silver nitrate, 5% potassium chromate.
The pressurizing pressure in the step 3) is 10MPa-100MPa, and more preferably 10MPa-50 MPa; the pressure dip dyeing time is 5-30 days. Most preferably, the pressurizing pressure is 15MPa, the complete morphological structure of the neuron can be impregnated only by pressurizing for 6 hours, and the brain tissue sample can not be impregnated under the normal pressure condition, so that the impregnating time of the brain tissue is greatly reduced.
The optimal padding time can be selected according to brain tissues of different animals and brain tissue slices with different thicknesses, for example, the optimal padding time for the whole brain hypertension of a mouse is 5 days, the optimal padding time for the whole brain hypertension of a rat is 10 days, and the optimal padding time for the whole brain hypertension of a new zealand white rabbit is 20 days.
The slicing equipment in the step 4) comprises a freezing slicer, a vibrating slicer and a paraffin slicer, and the thickness of the slices ranges from 1 μm to 5000 μm. Wherein, the optimal thickness for observation by an optical microscope is 100 μm; the thickness of the 1000-2000 μm section is selected for X-ray microscope imaging.
The staining reagent in the step 5) comprises: 10% -25% of ammonia water solution or 1% -10% of lithium hydroxide solution.
The staining time in the step 5) is 10min-24h or longer, and different staining times can be selected according to the thickness of the section, for example, the staining time of the section with the thickness of 100 μm is 10min, and the staining time of the whole brain of the mouse is 24 h.
The dehydration in the step 5) is performed by adopting a mode of gradient alcohol (30%, 50%, 70%, 85%, 90%, 95%, 100%) to perform brain tissue dehydration treatment, and the dehydration time of alcohol with each concentration is adjusted and optimized according to the thickness of the brain tissue slices. The tissue embedding mode can be resin embedding, paraffin embedding and the like.
And 6) imaging by using the optical microscope and observing by using the synchrotron radiation X-ray imaging, wherein the optical microscope refers to a microscope which can be used for optical imaging, such as a laser confocal microscope, a two-photon microscope, a lattice layer optical microscope, a super-resolution optical microscope and the like. The synchrotron radiation X-ray imaging observation refers to that brain tissue X-ray two-dimensional and three-dimensional imaging is carried out at synchrotron radiation X-ray related imaging line stations, such as an X-ray micron CT imaging line station, a nano CT imaging line station, a soft X-ray STXM imaging line station, a CDI imaging line station and the like, imaging data of brain nerve connection is obtained, and an imaging atlas is drawn. The resolution of X-ray micro CT is isotropic resolution of 0.3 μm, imaging resolution of nano CT is 20nm, soft X-ray STXM imaging resolution is 40nm, and CDI imaging resolution is 10 nm.
The positive progress effects of the invention are as follows: 1) the traditional Golgi staining method for brain tissue has been used for over one hundred years, and the research on the aspect of finding the initial application mainly in optical microscopic imaging has a prominent contribution to the establishment of the theory of mind. However, there are also some deficiencies with Golgi staining: low efficiency of dyeing, randomness and discontinuity of dyeing, unobvious dyeing of some tiny nerve fibers and the like. The method combines pressurization with the traditional Golgi dyeing method, promotes the permeation of dyeing liquid in nerve tissues and the distribution of dyeing liquid in small nerve fibers through high pressure, improves the marking efficiency of neurons, reduces the dyeing time and improves the contrast of the small nerve fibers under the X-ray irradiation, optimizes the traditional Golgi dyeing method by the new method, and establishes a novel Golgi dyeing technology suitable for the imaging of brain tissues by synchrotron radiation X-rays. 2) At present, the imaging method of the brain tissue mainly comprises an electron microscope and an optical microscope, but the imaging speed of the electron microscope is very slow, and large-scale imaging cannot be carried out. Since the resolution of a common optical microscope is limited by the diffraction limit, particularly the resolution in the Z-axis direction is low, it is difficult to break through 1 μm, and it is difficult to perform imaging analysis on fine nerve fibers (<1 μm). The imaging speed of the synchrotron radiation-based X-ray imaging technology is obviously superior to that of an electron microscope and an optical microscope, the X-ray imaging has isotropic high resolution, and the resolution in the Z-axis direction is also obviously superior to that of a common optical microscope, so that the synchrotron radiation X-ray imaging method has wide prospect when being applied to brain tissue imaging. However, limited by the development of staining technology, there are few reports of the application of synchrotron radiation X-ray imaging method to brain tissue imaging. The method provided by the invention can greatly expand the application of the synchrotron radiation X-ray imaging method in the aspect of brain tissue imaging.
In a word, the invention establishes a novel high-efficiency Golgi staining method for large-size brain tissues, optimizes the traditional Golgi staining method and has good brain imaging application prospect.
Drawings
FIG. 1A is an optical microscopic image of a cerebral cortical region after a mouse brain tissue is impregnated with a Golgi staining solution for 6 hours under normal atmospheric pressure (0.1MPa), FIG. 1B is an optical microscopic image of a cerebral cortical region after a mouse brain tissue is impregnated with a Golgi staining solution for 6 hours under lower pressurization (1.5MPa), and FIG. 1C is an optical microscopic image of a cerebral cortical region after a mouse brain tissue is impregnated with a Golgi staining solution for 6 hours under higher pressurization (15 MPa);
FIG. 2A is an enlarged view of pyramidal neurons in the cortical region of a mouse after 6h of padding with a Golgi staining solution under relatively low pressure (1.5MPa), and FIG. 2B is an enlarged view of pyramidal neurons in the cortical region of a mouse after 6h of padding with a Golgi staining solution under relatively high pressure (15 MPa);
FIG. 3A is an optical microscopic image of a cerebral cortical region after 24 hours of padding with a Golgi staining solution of mouse brain tissue under normal atmospheric pressure (0.1MPa), FIG. 3B is an optical microscopic image of a cerebral cortical region after 24 hours of padding with a Golgi staining solution of mouse brain tissue under lower pressurization (1.5MPa), and FIG. 3C is an optical microscopic image of a cerebral cortical region after 24 hours of padding with a Golgi staining solution of mouse brain tissue under higher pressurization (15 MPa);
FIG. 4A is an enlarged view of pyramidal neurons in the cortical region of a mouse after 24h of padding with Golgi staining solution under relatively low pressure (1.5MPa), and FIG. 4B is an enlarged view of pyramidal neurons in the cortical region of a mouse after 24h of padding with Golgi staining solution under relatively high pressure (15 MPa);
FIG. 5A is an optical microscopic image of the hippocampal region of the brain of a mouse after a prolonged immersion in a Golgi staining solution for 15 days under normal atmospheric pressure (0.1MPa), and FIG. 5B is an optical microscopic image of the hippocampal region of the brain of a mouse after a prolonged immersion in a Golgi staining solution for 15 days under a lower pressure (1.5 MPa).
Detailed Description
The present invention will be further described with reference to the following specific examples. It should be understood that the following examples are illustrative only and are not intended to limit the scope of the present invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.
The invention is exemplified by, but not limited to, mouse brain tissue, and the Golgi staining solution is potassium dichromate (K)2Cr2O7) Mercuric chloride (HgCl)2) Potassium chromate (K)2CrO4) The mass volume ratio of the dye is 5%, and the dyeing time is mainly not more than 15 days; the pressurizing pressure range is 1-20MPa, so as to avoid damaging the cell structure of the brain tissue; the slice is a frozen slice, the thickness of the slice is 100 μm by an optical microscope, and the thickness of the slice for synchrotron radiation X-ray imaging is 1 mm. The following examples are provided to illustrate the effects of the present invention, by combining the pressurization with the conventional Golgi staining to establish a novel Golgi staining method suitable for synchrotron radiation X-ray brain tissue imaging.
Example 1 pressure-facilitated Dip staining with Golgi staining solution of neuronal cell bodies and nerve fibers
Deeply anaesthetizing the mouse with 1% pentobarbital, removing blood by heart perfusion according to the conventional experiment, quickly taking out the whole brain of the mouse, and adding prepared Golgi staining solution (5% potassium dichromate (K)2Cr2O7) 5% mercuric chloride (HgCl)2) 5% potassium chromate (K)2CrO4) ) and applying different pressures for dyeing for 6 hours, wherein the control group is at normal atmospheric pressure (0.1MPa), the low pressure group is at 1.5MPa, and the high pressure group is at 15 MPa. And after dyeing is finished, taking out the brain tissue block, dehydrating the brain tissue block in a 30% sucrose solution at 4 ℃ in the dark for about 1 day, taking out the brain tissue block, and dehydrating the brain tissue block in a 30% sucrose solution at 4 ℃ in the dark for 1 day. In this example, the section was made by freezing section, the section was sagittal, the thickness was 100 μm, the cut mouse brain tissue piece was reacted in 20% ammonia water for 10 minutes, the reaction was stopped by washing with water, and the glycerol gelatin section was used for observation under a microscope.
FIGS. 1A to 1C show the staining patterns of neurons in cortical layer of mouse brain stained with Golgi staining solution for 6 hours under different pressures. As shown in fig. 1A, neurons in the cortical brain of the mouse were not substantially stained at normal pressure for 6 hours, and neuronal cells were not visible throughout the visual field. When pressurized at 1.5Mpa, the cell bodies of neurons and a part of nerve fibers were stained, but the staining of neuron fibers was not ideal, and only a few large branched structures were stained (fig. 1B). When a larger pressure of 15MPa is applied, the cell body and the fibers of the nerve fibers are impregnated, the fibers of the nerve fibers are completely dyed, the primary structure, the secondary structure, the tertiary structure and the like of most nerve fibers are also dyed (figure 1C), and the shape of the nerve fibers is relatively complete when the pressure of 15MPa is applied, so that the impregnation of the nerve fibers can be remarkably accelerated by high pressure.
FIGS. 2A-2B are enlarged views of individual neurons after Golgi staining of mouse brain tissue for 6 hours at two different pressures. FIG. 2A shows the morphology of pyramidal neurons in the cerebral cortex of mice under a compression of 1.5MPa, and the primary structure of the nerve fibers with large nuclei in the somatic neurons can be seen. When the pressure was increased to 15Mpa, the neuron morphology was relatively intact, and intact neuron dendritic morphology, and even dendritic spine morphology, could be seen (fig. 2B).
Example 2 increasing mouse cerebral cortex neuronal counts with increased staining efficiency
The mouse brain tissue treatment procedure and Golgi staining procedure were as in example 1. The mouse brain tissue is taken out and immediately placed into Golgi staining solution for dip-staining, wherein the control group is at normal atmospheric pressure (0.1MPa), the pressure of the low-pressure group is 1.5MPa, the pressure of the high-pressure group is 15MPa, and the dip-staining time is 24 hours. As shown in FIG. 3A, after 24h of Golgi staining solution staining of the brain tissue of the mice in the normal atmospheric pressure group, a large number of neuronal soma and large nerve fiber morphology in the cerebral cortex area of the mice can be observed under an optical microscope, but the overall neuronal morphology is still incomplete, and some small nerve fibers are not stained. After the mouse brain tissue is stained by the Golgi staining solution under the pressure of 1.5MPa for 24h, the complete morphology of the neuron cell bodies and nerve fibers can be seen under an optical microscope (figure 3B); when the applied pressure was further increased to 15Mpa, the morphology of the neurons was more intact and the number of neurons also increased significantly (fig. 3C).
FIG. 4 is a magnified view of a single neuron after 24 hours Golgi staining of mouse brain tissue at two different pressures. FIG. 4A shows the morphology of pyramidal neurons in the cerebral cortex of mice at a pressure of 1.5MPa, which is relatively intact, and a large number of primary structures of the cell bodies and dendrites of the neurons can be observed, and secondary structures of partial regions can also be observed (FIG. 4A). When the pressure was increased to 15Mpa, the neuron morphology was more complete, a large amount of primary and secondary structures of the neuron cell bodies and dendrites were observed, and a small amount of tertiary structures were observed in some regions (fig. 4B).
Example 3 Long-term compression of neurons in hippocampal region of mouse brain to complete morphology and clear visualization of large numbers of neuronal dendritic structures
The mouse brain tissue treatment process and the Golgi staining method were the same as in example 1. The mouse brain tissue is immediately placed into Golgi staining solution for dip-staining after being taken out, wherein the control group is subjected to dip-staining for 15 days under normal atmospheric pressure (0.1MPa) and the pressure of the pressurizing group is 1.5 MPa. As shown in FIG. 5A, after the mouse brain tissue is impregnated with Golgi staining solution at normal atmospheric pressure (0.1MPa) for 15 days, the neuron structure and morphology in the hippocampal region of the mouse brain are relatively intact; after the mouse brain tissue Golgi staining solution is soaked for 15 days under the condition of applying the pressure of 1.5Mpa, the neuron structure and the form of the hippocampal region of the mouse brain are more complete and clear, and a large number of typical clustered neurons can be observed (figure 5B).
The above embodiments are merely preferred embodiments of the present invention, which are not intended to limit the scope of the present invention, and various changes may be made in the above embodiments of the present invention. All simple and equivalent changes and modifications made according to the claims and the content of the specification of the present application fall within the protection scope of the claims of the present patent application. The invention has not been described in detail in order to avoid obscuring the invention.
Claims (10)
1. A method for efficient golgi staining of large-size brain tissue, comprising the steps of:
1) providing a pressurizing apparatus; 2) obtaining animal brain tissue; 3) preparing a Golgi staining solution, immersing the animal brain tissue in the Golgi staining solution, and pressurizing in a pressurizing device, wherein the pressurizing pressure is 1-100 MPa, and the pressurizing immersion time is 1-30 days; 4) slicing the animal brain tissue; 5) staining, dehydrating and embedding the sliced animal brain tissue; and 6) optical microscopy imaging and/or synchrotron radiation X-ray imaging.
2. The method for high efficiency Golgi staining of large size brain tissue according to claim 1, wherein the pressurizing means in step 1) comprises a gas pressurizing means and a liquid pressurizing means.
3. The method for high efficiency golgi staining of large size brain tissue according to claim 1, wherein the animal brain tissue in step 2) comprises: drosophila brain tissue, zebrafish brain tissue, mouse brain tissue, rat brain tissue, rabbit brain tissue, cat brain tissue, dog brain tissue, and monkey brain tissue.
4. The method for high efficiency Golgi staining of large brain tissue according to claim 1, wherein the animal brain tissue of step 2) comprises fresh unfixed animal brain tissue or animal brain tissue fixed with a fixative solution comprising: paraformaldehyde, glutaraldehyde, ethanol, methanol, glacial acetic acid, acetone, and formalin.
5. The method for high efficiency Golgi staining of large-sized brain tissue according to claim 1, wherein the composition of the Golgi staining solution in step 3) is: 1) 5% of potassium dichromate, 5% of mercury chloride and 5% of potassium chromate; or 2) 5% potassium dichromate, 1% silver nitrate, 5% potassium chromate.
6. The high efficiency golgi staining method for large size brain tissue according to claim 1, wherein the pressure in step 3) is 10MPa to 100MPa and the pressure dip staining time is 5 to 30 days.
7. The method for high efficiency Golgi staining of large size brain tissue according to claim 1, wherein the sectioning equipment used in step 4) comprises a cryomicrotome, a vibrating microtome and a paraffin microtome, and the thickness of the sections ranges from 1 μm to 5000 μm.
8. The method for high efficiency Golgi staining of large-sized brain tissue according to claim 1, wherein the staining reagent used in step 5) comprises: 10% -25% of ammonia water solution or 1% -10% of lithium hydroxide solution.
9. The method for high efficiency Golgi staining of large size brain tissue according to claim 1, wherein the staining time in step 5) is 10min-24 h.
10. The method for high efficiency Golgi staining of large size brain tissue according to claim 1, wherein the dehydration in step 5) is performed with gradient alcohol.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116429544A (en) * | 2023-03-31 | 2023-07-14 | 中国科学院上海高等研究院 | A Synchrotron Radiation X-ray Imaging Staining Method for Human Brain Tissue Imaging |
| CN119595603A (en) * | 2024-11-24 | 2025-03-11 | 北京工业大学 | RNA m based on antibody coupled DNA nanocluster probe6A modified single cell multi-mode analysis method |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1814756A (en) * | 2006-03-02 | 2006-08-09 | 武汉大学 | Method for preparing hard-tissue low-temperature buried cold-ester |
| CN103471898A (en) * | 2013-09-30 | 2013-12-25 | 合肥工业大学 | Rapid neuron staining method based on Golgi silver staining method |
| CN105164264A (en) * | 2012-12-12 | 2015-12-16 | 布罗德研究所有限公司 | Delivery, engineering and optimization of systems, methods and compositions for sequence manipulation and therapeutic applications |
| CN107727463A (en) * | 2017-09-18 | 2018-02-23 | 南方医科大学珠江医院 | The method that the preparation of super thick histotomy and cellular level reproduce tissue three-dimensional morphosis |
| CN107941587A (en) * | 2017-12-20 | 2018-04-20 | 华南师范大学 | A kind of method that improved Golgi Cox dyeing prepares brain tissue paraffin section |
| US20180306688A1 (en) * | 2017-04-21 | 2018-10-25 | Yeu-Kuang Hwu | Method and kit for staining neural tissue sample and method for visualizing neurons |
-
2020
- 2020-02-20 CN CN202010104757.0A patent/CN113281130B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1814756A (en) * | 2006-03-02 | 2006-08-09 | 武汉大学 | Method for preparing hard-tissue low-temperature buried cold-ester |
| CN105164264A (en) * | 2012-12-12 | 2015-12-16 | 布罗德研究所有限公司 | Delivery, engineering and optimization of systems, methods and compositions for sequence manipulation and therapeutic applications |
| CN103471898A (en) * | 2013-09-30 | 2013-12-25 | 合肥工业大学 | Rapid neuron staining method based on Golgi silver staining method |
| US20180306688A1 (en) * | 2017-04-21 | 2018-10-25 | Yeu-Kuang Hwu | Method and kit for staining neural tissue sample and method for visualizing neurons |
| CN107727463A (en) * | 2017-09-18 | 2018-02-23 | 南方医科大学珠江医院 | The method that the preparation of super thick histotomy and cellular level reproduce tissue three-dimensional morphosis |
| CN107941587A (en) * | 2017-12-20 | 2018-04-20 | 华南师范大学 | A kind of method that improved Golgi Cox dyeing prepares brain tissue paraffin section |
Non-Patent Citations (2)
| Title |
|---|
| GINA E. SOSINSKY ET AL.,: "The combination of chemical fixation procedures with high pressure freezing and freeze substitution preserves highly labile tissue ultrastructure for electron tomography applications", 《JOURNAL OF STRUCTURAL BIOLOGY》, no. 161, 31 December 2008 (2008-12-31) * |
| 王蕾;张芳;汤仁仙;张冠群;牛海晨;: "大鼠脑组织高尔基染色三种制备方法的比较", 临床与实验病理学杂志, no. 01, 17 January 2017 (2017-01-17) * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116429544A (en) * | 2023-03-31 | 2023-07-14 | 中国科学院上海高等研究院 | A Synchrotron Radiation X-ray Imaging Staining Method for Human Brain Tissue Imaging |
| CN119595603A (en) * | 2024-11-24 | 2025-03-11 | 北京工业大学 | RNA m based on antibody coupled DNA nanocluster probe6A modified single cell multi-mode analysis method |
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