Large-volume sample fluorescence labeling method based on tissue fixation
By generating autofluorescent labels on biological tissue samples using glyoxal fixative and combining this with laser scanning confocal microscopy, the problem of tissue damage in large samples in traditional techniques was solved, enabling rapid and uniform acquisition of three-dimensional structural information.
Patent Information
- Application Number
- CN202610159528.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-02-04
- Publication Date
- 2026-03-20
AI Technical Summary
Traditional histological research techniques cannot fully reveal the three-dimensional structural information of biological tissue samples, and existing three-dimensional histological research techniques are prone to causing damage to large tissue samples, making them unsuitable for the study of complete large tissue and organ samples.
After fixation with paraformaldehyde, biological tissue samples were placed in a fixative containing glyoxal. The autofluorescence generated by glyoxal fixation was used for fluorescent labeling, and three-dimensional structural information was obtained by laser scanning confocal microscopy.
It enables rapid and uniform fluorescent labeling of large tissue samples without defatting, reducing sample processing time and enabling the acquisition of structural information such as cell bodies, fiber bundles, and blood vessels. It is suitable for large and dense tissue samples.
Smart Images
Figure CN121702837A_ABST
Abstract
Description
Technical Field
[0001] This application relates to the field of biological tissue staining technology, and in particular to a method for fluorescent labeling of large-volume samples based on tissue fixation. Background Technology
[0002] Understanding how tissue structure changes from the cellular level to the global scale is a crucial pathway for next-generation biomedical research. It holds significant research value, particularly in fields such as neuroscience, immunology, and genetics. For example, it allows for the mechanistic elucidation of various aspects of neuronal development, including the formation of complex three-dimensional networks, the occurrence and metastasis of pathologies, and species development. Therefore, histological studies of intact tissues and organs, based on their three-dimensional structure, acquire information on different types of tissue structures to achieve objective and accurate identification of biological tissue samples. This has a profound impact on understanding the fundamental principles of life's structure and the mechanisms of disease.
[0003] Traditional histological studies involve immunofluorescence or hematoxylin and eosin (HE) staining after tissue sectioning. However, these techniques only achieve single-layer two-dimensional mapping and analysis. Two-dimensional tissue processing leads to incomplete tissue information, loss of spatial structural information, and an inability to effectively quantify biological information, failing to fully reveal the morphology and molecular composition of biological tissue samples. To obtain complete molecular structural information from biological tissue samples, an effective method is three-dimensional tissue research techniques. Current techniques for three-dimensional histological research primarily utilize optical transparency techniques combined with tissue transparency enhancement techniques. Optical transparency techniques mainly include fMOST (f-MOST), sequential two-photon computed tomography (STPT), fractional-scale microscopy (FAST), and light-sheet microscopy (LSM), all of which can achieve micrometer-level resolution reconstruction of tissues and organs. However, these techniques need to be combined with tissue transparency enhancement techniques, which mainly include tissue labeling and tissue transparency techniques. Tissue labeling primarily involves fluorescent molecules and immunofluorescence labeling. The main challenges of these techniques are the depth and uniformity of antibody or dye diffusion, and the tissue damage caused by prolonged processing of large samples, making them unsuitable for studying complete, large-sample tissues and organs.
[0004] To meet the needs of three-dimensional histology research, there is an urgent need to develop a new technique for large-sample tissue research. Summary of the Invention
[0005] The purpose of this application is to provide a novel fluorescent labeling method for large-volume samples based on tissue fixation.
[0006] The following technical solution is adopted in this application:
[0007] One aspect of this application discloses a method for fluorescent labeling of large-volume samples based on tissue fixation, comprising the following steps:
[0008] The sample fixation procedure includes fixing biological tissue samples with paraformaldehyde;
[0009] The fluorescent labeling step includes placing the fixed biological tissue sample in a fixative containing glyoxal, and using the autofluorescence generated by the browning caused by glyoxal fixation to achieve fluorescent labeling of the biological tissue sample.
[0010] The large-volume sample in this application refers to, for example, large-volume organs and tissues including but not limited to the whole brain, heart, liver, lungs, and kidneys.
[0011] It should be noted that this application's research found that the autofluorescence generated by browning during glyoxal fixation can effectively fluorescently label large samples of biological tissues, and has the following advantages: (i) easy to process; (ii) rapid penetration into tissue without producing artifacts such as diffusion rings; (iii) suitable for large and dense tissue samples; and (iv) uniform and complete staining. Compared with other existing molecular labeling technologies, the fluorescent labeling method of this application can not only achieve cell body and fiber bundle labeling without defatting, but also obtain vascular information through contrast; moreover, the method of this application is easy to process, and can rapidly and uniformly penetrate the sample within only 48 hours, greatly reducing sample processing time. In one embodiment of this application, the method of this application can not only obtain structural information of brain tissue, but also structural information of various organs such as the heart, kidneys, and liver.
[0012] Preferably, the concentration of glyoxal in the stationary solution containing glyoxal is not less than 9 wt%.
[0013] It should be noted that the concentration of glyoxal should not be too low, for example, not less than 9 wt%, otherwise it will easily lead to poor fluorescent labeling effect.
[0014] Preferably, the glyoxal-containing fixative includes glyoxal, acetic acid, and water, and the pH value is adjusted using an alkaline solution.
[0015] Preferably, the concentration of acetic acid is 8 v / v.
[0016] Preferably, the alkaline solution is NaOH.
[0017] Preferably, the pH value of the glyoxal-containing stationary solution is 4.1.
[0018] It should be noted that this application uses weak acids and bases such as acetic acid and NaOH to adjust the pH value, mainly to ensure that the pH value of the fixative containing glyoxal is 4.1. Under acidic conditions of pH=4.1, glyoxal can effectively promote the reaction rate with tissue.
[0019] Preferably, the fluorescent labeling step further includes placing the biological tissue sample in a fixative containing glyoxal and shaking it at 60°C for 48 hours.
[0020] It should be noted that this application involves fluorescent labeling after shaking treatment at 60°C for 48 hours. The reaction temperature should not be too high or too low; lower temperatures will slow down the reaction time, while higher temperatures will cause tissue shrinkage and significant deformation. Therefore, fluorescent labeling is preferably performed at 60°C. Similarly, the reaction time should not be too long or too short. Excessive time will result in severe browning of the tissue, and even with the use of a contrasting reagent, the background information cannot be effectively masked.
[0021] Preferably, the oscillation condition is 50-120 rpm.
[0022] Preferably, the fluorescent labeling step further includes rinsing with PBS buffer after shaking to remove excess fixative.
[0023] Preferably, the concentration of the PBS buffer is 0.01 mol / L.
[0024] Preferably, the sample fixation step further includes placing the biological tissue sample in a paraformaldehyde fixative and fixing it overnight at 4°C.
[0025] Preferably, in the sample fixation step, the fixative is 4% paraformaldehyde.
[0026] Preferably, the sample fixation step further includes rinsing with PBS buffer after fixation to remove excess fixative.
[0027] Preferably, the concentration of the PBS buffer is 0.01 mol / L.
[0028] Preferably, the method also includes storing the rinsed biological tissue samples in PBS buffer for later use.
[0029] Preferably, the PBS buffer is stored at a temperature of 4°C.
[0030] Another aspect of this application discloses a method for obtaining three-dimensional information of biological tissue samples, including fluorescent labeling of the biological tissue sample to be tested using the fluorescent labeling method of this application, and then obtaining the three-dimensional structural information of the biological tissue sample to be tested using laser scanning confocal microscopy and fluorescence microscopy optical section tomography.
[0031] Preferably, the biological tissue samples to be tested include, but are not limited to, the brain, heart, lungs, kidneys, and liver.
[0032] It should be noted that the three-dimensional information acquisition method of this application does not require fluorescent dyes and antibody labeling. By acquiring high-resolution imaging results of different regions, it can effectively obtain structural information such as cell bodies, fiber bundles, and blood vessels of large sample tissue structures. This method is not only applicable to whole brain tissue, but can also obtain three-dimensional structural information such as small arteries and venules in the heart, and can effectively obtain three-dimensional information such as bronchi and alveoli in lung tissue, glomeruli and vascular glomeruli in kidney tissue, and hepatic sinusoids in liver tissue. This opens up a new way for acquiring three-dimensional structural information of different large sample tissues and organs at the submicron scale.
[0033] The beneficial effects of this application are as follows:
[0034] The fluorescent labeling method of this application does not require fluorescent dyes and antibody labeling. It can not only label cell bodies and fiber bundles without defatting, but also obtain vascular information through contrast. Moreover, the method of this application is easy to process and can quickly and uniformly penetrate the sample, greatly reducing the sample processing time. It opens up a new way to obtain three-dimensional structural information of different large sample tissues and organs at the submicron scale. Attached Figure Description
[0035] Figure 1 These are the results of the gelatin permeation simulation experiment in the embodiments of this application;
[0036] Figure 2 These are the fluorescence intensity results of mouse brain tissue after fixation with different fixatives in the embodiments of this application;
[0037] Figure 3 This is the result of obtaining different structural information from mouse brain tissue through fixation and background removal processing in the embodiments of this application;
[0038] Figure 4 This is a three-color channel imaging and merging image of a large sample of tissues and organs from the C57 mouse brain in the embodiments of this application;
[0039] Figure 5 These are tissue sections and imaging results of the mouse heart, lungs, kidneys, and liver in the embodiments of this application;
[0040] Figure 6 These are images showing the HE and 9% glyoxal labeling results of mouse brain and kidney tissues in the embodiments of this application;
[0041] Figure 7 This is a three-dimensional structural information image of the C57 mouse kidney obtained by fMost in the embodiments of this application. Detailed Implementation
[0042] The present application will be further described in detail below through specific embodiments. The following embodiments are only for further explanation of the present application and should not be construed as limiting the present application.
[0043] Unless otherwise specified, all materials and reagents used in the following examples are commercially available, and all experimental methods used in the following examples are conventional methods.
[0044] Example
[0045] I. Main Reagents and Equipment
[0046] Preparation of 9% glyoxal (Gly) fixative: 22.5 mL of 40% glyoxal solution, 69.5 mL of H2O, 8 mL of acetic acid, and adjust the pH to 4.1 with 5 mol / L NaOH (about 8 mL).
[0047] Preparation of 4% PFA fixative: Add 10 mL of 40% paraformaldehyde fixative to 90 mL of 0.01 mol / L PBS to dilute to 4%.
[0048] Preparation of 4% PFA + 1% GA fixative: Use 25 mL of 16% PFA and 4 mL of 35% GA, and bring the volume to 100 mL with 0.01 mol / L PBS.
[0049] 0.01 mol / L PBS was purchased from Maclean's, catalog number: P854542-1EA. Mice were purchased from Spiefol (Beijing) Biotechnology Co., Ltd., strain: C57BL / 6J, age: 8 weeks, sex: male, weight: 17-19g. Peristaltic pump: Lange Constant Flow Pump Co., Ltd., model: BT100-3J. Air bath constant temperature shaker: Jiangsu Shenglan Instrument Manufacturing Co., Ltd., model: XMTE-206.
[0050] II. Methods and Results
[0051] Sampling: Animals underwent cardiac perfusion, followed by sequential perfusion with 0.01 mol / L PBS and 4% PFA fixative. Organs of interest were dissected and placed in 4% PFA fixative overnight at 4°C. The fixed organs were then rinsed in 0.01 mol / L PBS to remove excess fixative, and subsequently stored in 0.01 mol / L PBS at 4°C.
[0052] Fluorescent labeling treatment: Biological tissues were placed in 9% Glyoxal (glyoxal) fixative and shaken at 60°C for 48 hours. Then, excess fixative was washed off in 0.01 mol / L PBS solution.
[0053] Under these conditions, the method allows the 9% Gly fixative to undergo a Schiff base reaction with the tissue, which in turn leads to an oxidation reaction. This enables large samples of tissues and organs to be uniformly fixed in a short time, effectively achieving fluorescent labeling of cells, fibers, blood vessels, etc.
[0054] Three-dimensional information acquisition: Tissue structure information is acquired through laser scanning confocal microscopy (LSCM) and fluorescence optical section tomography (fMOST).
[0055] (1) Simulation experiment of aldehyde permeation gelatin
[0056] Fixed and defatted tissue samples resemble electrolyte gels; therefore, based on the molecular size and cross-linking characteristics of different aldehydes such as PFA, GA, and Gly, 10% gelatin was used to replace tissue organs for aldehyde permeability testing, and fixation changes in brain tissue were estimated. The specific testing method included: 10% gelatin was heated and poured into a mold to completely solidify (approximately 30 min), then placed in 4% PFA fixative, 4% PFA + 1% GA fixative, and 9% Gly fixative, respectively, and shaken at room temperature for 7 days. Quantitative analysis was then performed based on the color changes compared to the control group, as well as the depth of these color changes.
[0057] The test results are as follows Figure 1 As shown.
[0058] Figure 1 The results showed that the penetration depth and uniformity of 4% PFA fixative and 9% Gly fixative were much greater than those of 4% PFA + 1% GA fixative. Furthermore, the 9% Gly fixative turned brown to yellowish-brown during the long-term cross-linking process. This method can predict and reveal the changes in the penetration depth of aldehydes into brain tissue and the chemical changes of different aldehyde fixation.
[0059] (2) Fluorescent labeling of mouse brain tissue
[0060] Fluorescent labeling was performed using 4% PFA fixative, 4% PFA + 1% GA fixative, and 9% Gly fixative, respectively. Specifically, the whole brains of three mice were placed in 4% PFA fixative, 4% PFA + 1% GA fixative, and 9% Gly fixative, respectively, and shaken at 60°C for 48 hours. Excess fixative was then washed away in 0.01 mol / L PBS. Laser confocal microscopy (LSCM) was then used to image the brain tissue, and the results are shown below. Figure 2 As shown.
[0061] Figure 2The results showed that the background signal of the 4% PFA fixative, 4% PFA + 1% GA fixative, and 9% Gly fixative gradually increased. This phenomenon suggests that the browning of tissues after glyoxal fixation may be caused by further oxidation following the Schiff base reaction between the aldehyde and the amino group.
[0062] Mouse brain tissue fixed with 4% PFA and 9% Gly was imaged using LSCM. Images were acquired, and True Black, a background fluorescence reduction contrast agent, was used as a contrast agent. The results are as follows: Figure 3 As shown.
[0063] Figure 3 The results showed that fixation with 9% Gly effectively obtained information on the structure of mouse brain tissue.
[0064] This laid the foundation for more effective acquisition of large sample tissue structure information. We tried using True Black (purchased from Biotium, catalog number: 23007), a background fluorescence reduction contrast reagent. The method of using this reagent is as follows: the sample is treated with 9% glyoxal and rinsed with PBS. The tissue sample is then placed in 1 mL of 5% True Black solution (50 mL True Black + 950 mL 0.01 mol / PBS) and placed in a 37°C constant temperature shaker at 80 rpm for 12 hours.
[0065] The results showed that the use of this reagent can effectively obtain information about the structure of mouse brain tissue.
[0066] This novel tissue structure technique, which eliminates the need for fluorescent dyes and antibody labeling, opens up new avenues for acquiring three-dimensional structural information of large samples of different tissues and organs at the submicron scale.
[0067] (3) Large sample brain tissue and organ imaging of C57 mice
[0068] Using large samples of brain tissue and organs from C57 mice as the research subject, the specific methods included:
[0069] For sample fixation, large brain tissue samples from C57 mice were placed in 4% PFA fixative and fixed overnight at 4°C. After fixation, the large brain tissue samples from C57 mice were rinsed in 0.01 mol / L PBS solution to remove excess fixative.
[0070] The fluorescent labeling step involved placing a large sample of C57 mouse brain tissue in 9% Glyoxal (glyoxal) fixative and shaking it at 60°C for 48 hours. Excess fixative was then washed away in 0.01 mol / L PBS solution.
[0071] Then, LSCM was used to acquire three-channel images and merged images, as shown in the following figure. Figure 4 As shown. Figure 4 In the diagram, A shows the autofluorescence produced after browning of brain tissue fixed with 9% Gly; B shows autofluorescence signals in the blue, green, and red channels, with the green channel showing the strongest fluorescence signal; C and G show the magnified areas of the cortex in A, where cell body signals can be effectively obtained; D and H show the magnified areas of the white matter in A, where cell body and fiber bundle information can be effectively obtained; E and I show the magnified areas of the striatum in A, where cell body and fiber bundle information can be effectively obtained; F and J show the magnified areas of the SI region in A, where cell body and nerve fiber information can be effectively obtained.
[0072] Figure 4 The results showed that the fluorescent markers were consistent across different channels; the green channel exhibited the strongest fluorescence signal. High-resolution imaging of different regions demonstrated that this method can effectively acquire structural information such as cell bodies, fiber bundles, and blood vessels in large tissue samples, including the mouse brain.
[0073] (4) Fluorescent labeling of different organs
[0074] Using the same method as in "(3) Large Sample Tissue and Organ Imaging of C57 Mouse Brain", the heart, lung, kidney, and liver of the mice were fluorescently labeled in this case. Sampling: The mice underwent cardiac perfusion, sequentially perfused with 0.01 mol / L PBS and 4% PFA fixative. The heart, lung, kidney, and liver were then dissected and obtained. The following procedures were then performed on each organ:
[0075] The sample fixation procedure involves placing biological tissues and organs in 4% PFA fixative and fixing them overnight at 4°C. After fixation, large samples of tissues and organs are rinsed in 0.01 mol / L PBS solution to remove excess fixative.
[0076] The fluorescent labeling procedure involved placing large samples of tissues and organs, after fixation, in 9% Glyoxal (glyoxal) fixative and shaking at 60°C for 48 hours. Excess fixative was then washed away in 0.01 mol / L PBS. Finally, LSCM was used to acquire three-channel imaging and composite images, as shown below. Figure 5 As shown. Figure 5 In the diagram, image A is a slice of myocardial tissue, and the image below image A shows small arteries and venules; image B is a slice of lung tissue, and the image below image B shows bronchi and alveoli; image C is a slice of kidney tissue, and the image below image C shows glomeruli and glomeruli; image D is a slice of liver tissue, and the image below image D shows hepatic sinusoids.
[0077] Figure 5Figure A shows that myocardial tissue sections can effectively obtain arterioles and venules; Figure B shows that lung tissue sections can effectively obtain bronchi and alveoli; Figure C shows that kidney tissue sections can effectively obtain glomeruli and glomeruli; and Figure D shows that liver tissue sections can effectively obtain hepatic sinusoids. Therefore, the 9% Gly fixation method can effectively obtain structures of other tissues and organs besides mouse brain tissue, including arterioles and venules in mouse heart, bronchi and alveoli in mouse lung tissue, glomeruli and glomeruli in mouse kidney tissue, and hepatic sinusoids in mouse liver tissue.
[0078] Figure 6 Figures A and C show HE staining of mouse brain tissue. Figures B and D show the fluorescence labeling of mouse brain tissue using the 9% Gly fixation method to obtain the green and far-infrared channels. By comparing adjacent 4µm samples of the same sample labeled using different methods, it can be seen that the fluorescence achieved by the 9% Gly fixation method can achieve a similar HE labeling effect. Similarly, Figures E and G show HE staining of mouse kidney tissue, and Figures F and H show the fluorescence labeling of mouse kidney tissue using the 9% Gly fixation method to obtain the green and far-infrared channels, also achieving a similar HE labeling effect. Furthermore, detailed images from Figures B, D, F, H, J, L, N, and P provide information on the fibrous and cell body structures in mouse organ tissues for comparison due to HE staining.
[0079] Three-dimensional structural information of C57 mouse kidneys obtained by fMost is as follows: Figure 7 As shown, it includes information on tissue structures such as blood vessels, veins, glomeruli, and proximal convoluted tubules.
[0080] The results above show that, compared to other molecular marker techniques, the method presented in this example can not only label cell bodies and fiber bundles without defatting, but also obtain vascular information through contrast. This method is easy to process, rapidly and uniformly penetrating the sample within just 48 hours, significantly reducing sample processing time. This technique can obtain structural information not only from brain tissue, but also from various organs such as the heart, lungs, kidneys, and liver.
[0081] The above description, in conjunction with specific embodiments, provides a further detailed explanation of this application and should not be construed as limiting the specific implementation of this application to these descriptions. Those skilled in the art to which this application pertains can make several simple deductions or substitutions without departing from the concept of this application.
Claims
1. A method for fluorescent labeling large-volume samples based on tissue fixation, characterized in that: Includes the following steps, The sample fixation procedure includes fixing biological tissue samples with paraformaldehyde; The fluorescent labeling step includes placing the fixed biological tissue sample in a fixative containing glyoxal, and using the autofluorescence generated by the browning caused by glyoxal fixation to achieve fluorescent labeling of the biological tissue sample.
2. The fluorescent labeling method according to claim 1, characterized in that: The concentration of glyoxal in the fixative containing glyoxal is not less than 9 wt%.
3. The fluorescent labeling method according to claim 2, characterized in that: The glyoxal-containing fixative includes glyoxal, acetic acid, and water, and the pH value is adjusted using an alkaline solution. Preferably, the concentration of the acetic acid is 8 v / v%. Preferably, the alkaline solution is NaOH; Preferably, the pH value of the glyoxal-containing fixative is 4.
1.
4. The fluorescent labeling method according to any one of claims 1-3, characterized in that: The fluorescent labeling step further includes placing the biological tissue sample in a fixative containing glyoxal and shaking it at 60°C for 48 hours. Preferably, the oscillation condition is 50-120 rpm.
5. The fluorescent labeling method according to claim 4, characterized in that: The fluorescent labeling step further includes rinsing with PBS buffer after shaking to remove excess fixative. Preferably, the concentration of the PBS buffer is 0.01 mol / L.
6. The fluorescent labeling method according to any one of claims 1-3, characterized in that: The sample fixation step further includes placing the biological tissue sample in a paraformaldehyde fixative and fixing it overnight at 4°C.
7. The fluorescent labeling method according to claim 6, characterized in that: In the sample fixation step, the fixative is 4% paraformaldehyde.
8. The fluorescent labeling method according to claim 6, characterized in that: The sample fixation step also includes rinsing with PBS buffer after fixation to remove excess fixative. Preferably, the concentration of the PBS buffer is 0.01 mol / L; Preferably, the method also includes storing the rinsed biological tissue samples in PBS buffer for later use. Preferably, the PBS buffer is stored at a temperature of 4°C.
9. A method for acquiring three-dimensional information of a biological tissue sample, characterized in that: The method includes fluorescently labeling the biological tissue sample to be tested using the fluorescent labeling method described in any one of claims 1-8, and then obtaining the three-dimensional structural information of the biological tissue sample to be tested using laser scanning confocal microscopy and fluorescence microscopy optical section tomography.
10. The three-dimensional information acquisition method according to claim 9, characterized in that: The biological tissue samples to be tested include, but are not limited to, the brain, heart, lungs, kidneys, and liver.