Transparent reagent for clinical tissues and animal organs and transparent imaging method

Abstract

The invention discloses a transparentizing reagent for clinical tissues and animal organs and a transparentizing imaging method. The transparent reagent is a mixed reagent formed by dissolving 1-ethylimidazole and 3-[3-(cholamidopropyl) dimethylamino] propanesulfonic acid inner salt (CHAPS) in distilled water; wherein the mass volume ratio of the 1-ethylimidazole to the distilled water is 5%-40%, and the volume ratio of the CHAPS to the distilled water is 5%-25%. The pretreated biological tissue is soaked in a transparentizing reagent, and tissue transparentizing is achieved. The transparent reagent disclosed by the invention can be used for effectively and rapidly transparentizing clinical tissues, and is fluorescence-friendly and wide in applicability.

Description

Technical Field

[0001] The invention belongs to the field of biological sample tissue processing, and in particular relates to a transparentizing reagent for clinical tissues and animal organs and a transparentizing imaging method. Background Art

[0002] Tissue optical transparency technology uses chemical reagents to treat biological tissues, change the refractive index inside the biological tissues, thereby increasing the penetration of light into the biological tissues, and then increasing the imaging depth of the optical microscope on the biological tissues, which is conducive to the three-dimensional reconstruction of large-volume biological tissues and the analysis of neural circuits. At present, the most commonly used technical routes for optical transparency technology can be divided into four categories: the first is the simple immersion type, which has simple reagent components, but the transparency effect is often poor. The second is the organic solvent type, which uses organic solvents to dehydrate and match the refractive index of biological tissues. This type of method often has a better transparency effect, but it causes serious damage to biological tissues. The third is the hydration type, which generally has better protection for biological tissues and good fluorescence compatibility. The fourth is the hydrogel type, which can usually achieve good transparency, but due to the use of surfactants, the biological tissues will swell severely, and the cell membrane structure will be completely destroyed. In addition, the surfactant penetrates into the tissue very slowly, and the technical cost required for transparency is high. Summary of the invention

[0003] The purpose of the present invention is to provide a clinical tissue clearing reagent and its application which is convenient to use, fast in speed and effective for clinical tissues.

[0004] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0005] 1. A clearing reagent for clinical tissues and animal organs

[0006] The main reagent components of the transparent reagent include 1-ethylimidazole and 3-[3-(cholamidopropyl)dimethylamino]propanesulfonic acid inner salt (CHAPS), the mass volume ratio of the 1-ethylimidazole to distilled water is 5% to 40%; the mass volume ratio of the CHAPS to distilled water is 5% to 25%.

[0007] Preferably, the mass volume ratio of the 1-ethylimidazole to distilled water is 25%; the mass volume ratio of the CHAPS to distilled water is 10%.

[0008] The CHAPS and 1-ethylimidazole are both commercially available products and are the main transparent components, which function to remove heme and dissolve lipids.

[0009] 2. A method for clearing clinical tissues and animal organs, comprising:

[0010] The isolated clinical tissues and animal organs were pre-processed.

[0011] The pretreated biological tissue is immersed in the above-mentioned clearing agent.

[0012] The pretreatment includes one or more of tissue fixation, tissue sectioning, immunostaining, and chemical dye staining.

[0013] 3. Application of a clearing agent for clinical tissues and animal organs in biological tissue clearing and imaging

[0014] Beneficial effects of the present invention:

[0015] 1) The transparentizing agent used in the present invention has simple ingredients and can be flexibly selected according to different scenarios;

[0016] 2) The method of the present invention is simple and easy to implement, and has wide applicability;

[0017] 3) Compared with published tissue clearing reagents, the reagent proposed in the present invention can shorten the sample processing time. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a comparison diagram of a 500 micron thick clinical kidney before treatment with a reagent, after treatment with a transparent reagent, and after treatment with a refractive index matching reagent in Example 1 of the present invention;

[0019] Figure 2 This is a comparison diagram of mouse kidney before reagent treatment, after transparent reagent treatment, and after refractive index matching reagent treatment in Example 2 of the present invention;

[0020] Figure 3 This is a light sheet microscope image of the mouse kidney after reagent treatment in Example 2 of the present invention;

[0021] Figure 4 This is a comparison diagram of a 500 micron thick clinical human brain slice before and after reagent treatment in Example 3 of the present invention;

[0022] Figure 5 This is a confocal microscope image of a 500-micron-thick clinical human brain slice after reagent treatment in Example 3 of the present invention. DETAILED DESCRIPTION

[0023] The technical solution of the present invention is fully described below in conjunction with the accompanying drawings and embodiments.

[0024] The present invention provides a transparent reagent for clinical tissues and animal organs, comprising 1-ethylimidazole and CHAPS, wherein the mass volume ratio of the 1-ethylimidazole to distilled water is 5% to 40%; the mass volume ratio of the CHAPS to distilled water is 5% to 25%. Preferably, the volume ratio of the 1-ethylimidazole to distilled water is 15% to 30%, and the mass volume ratio of the CHAPS to distilled water is 10% to 20%. More preferably, the volume ratio of the 1-ethylimidazole to distilled water is 20%, and the mass volume ratio of the CHAPS to distilled water is 15%.

[0025] Among them, the chemical reagents involved in the present invention are all commercially available without special restrictions.

[0026] The present invention also provides a method for clearing clinical tissues and animal organs, comprising immersing the pretreated biological tissue in the clearing reagent.

[0027] The biological tissue may be derived from ex vivo human tissue and mouse organ, including brain, heart, liver, pancreas, lung, intestine, but not limited to the above.

[0028] The pretreatment includes one or more of tissue fixation, tissue sectioning, immunostaining, chemical dye staining, and virus labeling. Tissue fixation is preferably obtained by cardiac perfusion, using 1x PBS and 4% PFA to perfuse the animal in sequence, and after the perfusion is completed, the biological tissue is taken out, stored in 4% PFA, and stored in a refrigerator at 4 degrees. Tissue sectioning is preferably performed by oscillating sectioning, frozen sectioning, etc.

[0029] The biological tissue is immersed in the above-mentioned transparent reagent, specifically: incubated in a shaker at room temperature until transparent, preferably, in a shaker at 37 degrees Celsius until transparent. The incubation time varies according to the size of the biological tissue. For a 500 micron clinical kidney slice, the incubation time is preferably 4 hours.

[0030] Imaging of the present invention includes, but is not limited to, light sheet microscopy, confocal microscopy, two-photon microscopy, and wide-field microscopy.

[0031] In order to make the purpose, technical solutions and advantages of this patent more clear, this patent is further described in detail in combination with the embodiments below. It should be understood that the specific embodiments described here are only used to explain this patent and are not used to limit this patent. In addition, the technical features involved in each embodiment of this patent described below can be combined with each other as long as they do not conflict with each other.

[0032] Example 1: Clearing of clinical kidney sections

[0033] The 500-micron-thick clinical kidney tissue used in the experiment was obtained from the Second Affiliated Hospital of Zhejiang University. The specific steps are as follows: The clinical kidney tissue was photographed. Figure 1 As shown in the left figure in the figure. Then, the tissue was treated with a clearing agent (wherein the mass volume ratio of 1-ethylimidazole to distilled water was 10%; the mass volume ratio of CHAPS to distilled water was 10%) for 4 hours. After the clearing agent treatment was completed, the treated clinical kidney tissue was photographed, and the resulting photos are shown in the figure below. Figure 1 Finally, the slices were transferred to a refractive index matching solution, where the refractive index matching solution was FOCM (sorbitol, urea, and glycerol dissolved in dimethyl sulfoxide at a mass volume ratio of 20%, 30%, and 5%, respectively). The processed clinical kidney tissue was photographed, and the resulting photos are shown in the figure below. Figure 1 Right picture in .

[0034] Example 2: Clearing and imaging of mouse kidney organoids

[0035] Transparent mouse kidney tissue was prepared as follows. All animal experiments described in this description were performed in accordance with the guidelines of the Animal Ethics Committee of Zhejiang University, China. The specific steps are as follows: adult mice (8 weeks old) were deeply anesthetized with 1% pentobar, the chest cavity was opened, 40 ml of ice-cold PBS (phosphate buffered saline) was perfused through the heart to rinse the blood, and then 20 ml of 4% PFA by mass volume was perfused for fixation. The chest cavity was then opened, the kidney tissue was removed, placed in a test tube containing 4% PFA, and placed in a 4°C refrigerator overnight.

[0036] The mouse kidneys were photographed, and the pictures obtained were as follows Figure 2 As shown in the left figure in the figure. Then, a clearing agent (wherein the mass volume ratio of 1-ethylimidazole to distilled water is 20%; the mass volume ratio of CHAPS to distilled water is 5%) was added to the kidney for 3 days. After the clearing agent treatment was completed, the treated mouse kidney was photographed, and the resulting photo is shown in the figure below. Figure 2 As shown in the middle figure.

[0037] After the clearing agent treatment, immunofluorescence staining was performed. The anti-NPSH 1 first antibody was dissolved in the staining solution at a ratio of 1:100 (the volume ratio of dimethyl sulfoxide and donkey serum in PBS was 5% and 3%, respectively). The mouse kidney treated with the clearing agent was immersed in the staining solution and placed in a 37-degree shaker for incubation for 5 days. After the incubation was completed, the staining solution was aspirated with a pipette, replaced with PBS and placed in a 37-degree shaker for incubation for 1 hour, and then the PBS was replaced again for a total of 3 times. Then the second antibody was stained, and the corresponding second antibody was dissolved in the staining solution at a ratio of 1:100. The mouse kidney organ was placed in the second antibody for incubation in a 37-degree shaker for 5 days. After the incubation was completed, the liquid in the 24-well plate was aspirated with a pipette, 1xPBS was added, and it was placed in a 37-degree shaker for washing for 1 hour, and then the PBS was replaced again for a total of 3 times.

[0038] After immunofluorescence staining, the mouse kidneys were transferred to a refractive index matching solution, where the refractive index matching solution was CUBIC-R (antipyrine, nicotinamide, and N-butyldiethanolamine dissolved in water in a mass volume ratio of 45%, 30%, and 0.5%, respectively). After the mouse kidneys sank to the bottom, they were photographed. Figure 2 As shown in the right figure in the figure. The fluorescent image obtained by imaging the transparent mouse kidney using light sheet microscopy is shown in the figure below. Figure 3 as shown in .

[0039] Example 3: Clearing and imaging of human brain tissue

[0040] The 500-micron-thick clinical human brain tissue used in the experiment was obtained from the Chinese Brain Bank of the School of Medicine, Zhejiang University. Figure 4 As shown in the left figure in the figure, the tissue was then treated with a clearing agent (wherein the mass volume ratio of 1-ethylimidazole to distilled water was 25%; the mass volume ratio of CHAPS to distilled water was 10%) for 2 days.

[0041] After the treatment with the clearing agent, immunofluorescence staining was performed. The anti-CD31 first antibody was dissolved in the staining solution at a ratio of 1:100 (the volume ratio of dimethyl sulfoxide and donkey serum in PBS was 10% and 3%, respectively). The human brain tissue treated with the clearing agent was immersed in the staining solution and placed in a 37-degree shaker for incubation for 3 days. After the incubation was completed, the staining solution was aspirated with a pipette, replaced with PBS and placed in a 37-degree shaker for incubation for 1 hour, and then the PBS was replaced again for a total of 3 times. Then the second antibody was stained, and the corresponding second antibody was dissolved in the staining solution at a ratio of 1:100. The human brain slice was placed in the second antibody for incubation in a 37-degree shaker for 3 days. After the incubation was completed, the liquid in the 24-well plate was aspirated with a pipette, 1x PBS was added, and it was placed in a 37-degree shaker for washing for 1 hour, and then the PBS was replaced again for a total of 3 times.

[0042] After immunofluorescence staining, the human brain tissue was transferred to a refractive index matching solution, where the refractive index matching solution was CUBIC-R (antipyrine, nicotinamide, and N-butyldiethanolamine dissolved in water in a mass volume ratio of 45%, 30%, and 0.5%, respectively). After the human brain tissue sank to the bottom, a photo was taken. Figure 4 As shown in the right picture in the figure. The human brain tissue after being transparent was imaged using a confocal microscope, and the resulting fluorescent image is shown in Figure 5 as shown in .

[0043] Depend on Figure 1 , Figure 2 , Figure 4 It can be seen that the transparentizing reagent of the present invention can be used to treat biological tissues, clinical tissues, animal organs and other biological tissues to achieve tissue transparency. Figure 3 and Figure 5 It can be seen that by treating biological tissues with the transparentizing reagent of the present invention, imaging of biological tissues such as clinical tissues and animal organs can be achieved.

[0044] It is easy for technicians in this field to understand that the above description is only a preferred implementation method of this patent and is not intended to limit this patent. Any modifications made within the spirit and principles of this patent, which are equivalent to replacements and improvements, should be included in the scope of protection of this patent.

Claims

1. A clearing reagent for clinical tissues and animal organs, characterized in that: The transparentizing agent is a mixed agent formed by dissolving 1-ethylimidazole and CHAPS in distilled water.

2. The clinical tissue and animal organ clearing reagent according to claim 1, characterized in that: The mass volume ratio of the 1-ethylimidazole to distilled water is 5%-40%; the mass volume ratio of CHAPS to distilled water is 5%-25%.

3. The clinical tissue and animal organ clearing reagent according to claim 2, characterized in that: The mass volume ratio of the 1-ethylimidazole to distilled water is 15%-30%; the mass volume ratio of CHAPS to distilled water is 10%-20%.

4. A method for tissue clearing using the clearing agent according to any one of claims 1 to 3, characterized in that: Soak the pretreated biological tissue in a clearing agent to achieve clearing.

5. The tissue clearing method according to claim 4, characterized in that: The pretreatment includes one or more methods of tissue fixation, tissue sectioning, immunostaining, chemical dye staining, and virus labeling.

6. The tissue clearing method according to claim 5, characterized in that: The specific steps include: 1) Tissue fixation: obtaining biological tissues through cardiac perfusion; 2) Tissue sectioning: Tissue sections are obtained by oscillating sectioning or freezing sectioning; 3) Treatment with transparent reagent: adding transparent reagent to biological tissue; 4) performing immunofluorescence staining on the biological tissue after treatment with the clearing agent; 5) Transfer the immunostained biological tissue into a refractive index matching solution and then perform tissue imaging.

7. The tissue clearing method according to claim 4, characterized in that: The biological tissue includes clinical tissue or animal organ after ex vivo treatment.

8. Use of a clearing agent for clinical tissues and animal organs in clearing imaging, characterized in that: The transparentizing reagent according to any one of claims 1 to 3 is used to perform transparentizing treatment on the in vitro biological tissue, and the treated biological tissue is imaged.

9. Use of the clearing agent for clinical tissues and animal organs in clearing imaging according to claim 8, characterized in that: The imaging method includes imaging by light sheet microscopy, confocal microscopy, two-photon microscopy or wide-field microscopy.

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