CN115791339A - Method for transparentizing large-volume biological tissue sample - Google Patents
Method for transparentizing large-volume biological tissue sample Download PDFInfo
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- CN115791339A CN115791339A CN202310048015.4A CN202310048015A CN115791339A CN 115791339 A CN115791339 A CN 115791339A CN 202310048015 A CN202310048015 A CN 202310048015A CN 115791339 A CN115791339 A CN 115791339A
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Abstract
The invention belongs to the technical field of biological tissue transparentization, and relates to a transparentization method for a large-volume biological tissue sample, which comprises the following steps: (1) fixing; (2) decoloring; (3) degreasing; (4) dehydrating; (5) tissue transparentization; wherein in the step (3), the reagent used for degreasing is: the polyether compound is obtained by compounding polyether substances and salt. The transparentizing method provided by the invention realizes the rapid and complete transparentizing treatment of the large-volume sample of the biological tissue and overcomes the defects of other oily reagent transparentizing technologies.
Description
Technical Field
The invention belongs to the technical field of biological tissue transparentization, and relates to a transparentization method for a large-volume sample of biological tissue.
Background
With the continuous development of biomedicine, the three-dimensional reconstruction of tissue structures plays an increasingly important role in the research of tissue morphology and physiological pathology. Common medical imaging techniques such as Computed Tomography (CT), magnetic Resonance Imaging (MRI), etc. can reveal three-dimensional anatomical structures of living beings, but it is difficult to obtain tissue structures at a cellular resolution level. Modern optical section microscopic imaging technologies such as confocal and two-photon microscopes can realize three-dimensional imaging of tissues without physical sections, and the imaging depth can reach dozens to hundreds of micrometers. However, thick tissue layers have many non-uniform refractive indices of the internal material, and complete imaging of large volumes of tissue remains challenging. Bulk samples of biological tissue are opaque and, if it is desired to view the tissue cell structures within them, it is conventional to slice them into slices, such as paraffin sections, frozen sections, etc., and place the slices in a microscopic optical system for imaging.
The tissue transparentizing technology which is developed in recent years is a method of soaking by using a chemical reagent to enable tissues to become transparent, and a complete tissue three-dimensional stereo imaging is realized by using a novel optical imaging tool, such as a light-sheet microscope, without cutting the tissues. Various tissue transparentization technologies are rapidly developed, and at the present stage, the transparentization technologies are mainly classified into 3 types: hydrophilic tissue transparentizing technology, hydrophobic tissue transparentizing technology and hydrogel embedding method. And correspondingly, the clearing reagent is divided into: water-soluble reagents, oily reagents, hydrogel reagents.
Research literature proves that the degreasing agent is one of causes of primary immunofluorescence loss of the tissue sample, while most of the methods disclosed in the prior art adopt alcohols and esters as the degreasing agent, so that the immunofluorescence loss is caused, and the problems of heavy autofluorescence and large optical imaging background noise of the tissue sample after transparentization treatment exist.
Disclosure of Invention
The invention aims to provide a method for transparentizing a large-volume biological tissue sample.
In one aspect, the present invention provides a method for transparentizing a large volume sample of biological tissue, comprising the following steps:
(1) Fixing;
(2) Decoloring;
(3) Degreasing;
(4) Dehydrating;
(5) Clearing the tissue;
wherein in the step (3), the reagent used for degreasing is: the polyether substance and the corresponding salt are compounded to obtain the compound; the compounding ratio of the polyether substance to the salt is as follows in terms of v/v: 1:0.2 to 10.
Research literature proves that the degreasing agent is one of causes of primary immunofluorescence loss of the tissue sample, while most of the methods disclosed in the prior art adopt alcohols and esters as the degreasing agent, so that the immunofluorescence loss is caused, and the problems of heavy autofluorescence and large optical imaging background noise of the tissue sample after transparentization treatment exist. The invention adopts ethers as degreasing agents and compounds with surfactants, thereby improving the problems of primary immunofluorescence loss of tissue samples, heavy autofluorescence of tissue samples after transparentization treatment, large optical imaging background noise and the like.
In some embodiments, the compounding ratio of the polyether substance to the salt is as follows: 1:0.2 to 6; preferably, the compounding ratio of the polyether substance to the salt is as follows in terms of v/v: 1:4 to 6.
In some embodiments, the reagent used for degreasing has a concentration of 0.2% to 15% in v/v; preferably, the concentration of the reagent for degreasing is 1% -10%.
In some embodiments, the reagents used for defatting include, in v/v: 0.1-9% of polyether substance and 0.1-9% of salt; preferably, the reagents used for degreasing comprise: 1 to 5 percent of polyether substance and 1 to 5 percent of salt; preferably, the reagents used for degreasing comprise: 1 to 5 percent of polyether substance and 1 to 5 percent of salt; preferably, the reagents used for degreasing comprise: 1% of polyether substance and 5% of salt.
In some embodiments, the polyether material is selected from any one or more of fatty alcohol polyoxyethylene ether, alkylphenol polyoxyethylene ether, castor oil polyoxyethylene ether and lauric acid polyoxyethylene ether.
In some embodiments, the salt is selected from any one or more of sodium fatty alcohol-polyoxyethylene ether sulfate, sodium lauryl sulfate, and triethanolamine.
In some embodiments, in the step (3), the degreasing step comprises: adding a reagent for degreasing into the decolored tissue sample, and placing the tissue sample in a shaking table for degreasing; wherein the degreasing times are as follows: is more than or equal to 1 time.
In some embodiments, the number of defatting times is: 1 to 2 times; preferably, the number of degreasing times is: 2 times.
In some embodiments, the rotational speed of the shaker is from 40 to 60 rpm; the temperature is as follows: 35-40 ℃; preferably, the rotational speed of the shaker is 50 revolutions per minute; the temperature is as follows: at 37 ℃.
In some embodiments, the time of degreasing is: 20 to 30h; preferably, the degreasing time is as follows: and (5) 24h.
In some embodiments, the defatting reagent is added in a 20:1.
in some embodiments, in step (1), the reagents used for the immobilization are: 15 to 20 percent of paraformaldehyde solution; preferably, the fixed time is: 12 to 24 hours.
In some embodiments, in step (2), the reagents used for decolorization include, in w/v: 1 to 40 percent of ethylenediamine, 0.1 to 10 percent of sodium hydroxide and the balance of purified water; preferably, the reagents used for decolorization include: 10 to 30 percent of ethylenediamine, 0.1 to 5 percent of sodium hydroxide and the balance of purified water.
In some embodiments, the step of decolorizing comprises: adding the reagent for decoloring into the fixed tissue sample, and putting the fixed tissue sample in a shaking table for decoloring; preferably, the rotating speed of the shaking table is 40 to 60 revolutions per minute; the temperature is as follows: 35-40 ℃; preferably, the time of the decolorization is: 20 to 30h; preferably, the ratio of the added amount of the reagent for decoloring to the volume of the tissue sample is 20:1.
in some embodiments, in step (4), the dehydrated reagent comprises a first dehydrated reagent, a second dehydrated reagent.
In some embodiments, the first dehydration reagent comprises, in v/v: ethanol water solution with different concentration gradients; the second dehydrating reagent comprises, in v/v: absolute ethanol and xylene.
In some embodiments, the first dehydration reagent comprises, in v/v: 40-60% ethanol water solution, 60-80% ethanol water solution and 80-95% ethanol water solution; preferably, the first dehydrating reagent comprises: 50% -55% ethanol water solution, 65% -75% ethanol water solution and 85% -95% ethanol water solution.
In some embodiments, the second dehydration reagent comprises, in v/v: 10 to 90 percent of absolute ethyl alcohol and 90 to 10 percent of dimethylbenzene; preferably, the second dehydrating reagent comprises: 40 to 60 percent of absolute ethyl alcohol and 60 to 40 percent of dimethylbenzene.
In some embodiments, the step of dehydrating comprises:
1) Sequentially adding the first dehydration reagent into the tissue sample after the degreasing treatment, and placing the tissue sample in a shaking table for primary dehydration treatment;
2) Adding the second dehydration reagent into the tissue sample after the primary dehydration treatment in the step 1), and placing the tissue sample in a shaking table for secondary dehydration treatment.
In some embodiments, in the primary dewatering treatment, the rotation speed of the shaker is 40 to 60 rpm; the temperature is as follows: 35-40 ℃; preferably, the rotational speed of the shaker is 50 revolutions per minute; the temperature is as follows: at 37 ℃.
In some embodiments, the time of dehydration is: 6 to 15h; preferably, the dehydration time is: 6 to 12h;
in some embodiments, the ratio of the amount of the first decolorizing reagent added to the volume of the tissue sample is 20:1.
in some embodiments, the dehydration time of the ethanol aqueous solution with different concentration gradients in the first dehydration reagent is 2 to 5 hours respectively; preferably, the dehydration time of the ethanol aqueous solution with different concentration gradients in the first dehydration reagent is 2 to 4 hours respectively.
In some embodiments, in the secondary dewatering treatment, the rotational speed of the shaker is 40 to 60 revolutions per minute; the temperature is as follows: 35-40 ℃; preferably, the rotational speed of the shaker is 50 revolutions per minute; the temperature is as follows: at 37 ℃.
In some embodiments, the time for dehydration is: 20 to 30h; preferably, the dehydration time is: and (5) 24h.
In some embodiments, the ratio of the amount of the first decolorizing reagent added to the volume of the tissue sample is 20:1.
in some embodiments, in step (5), the agent for tissue clearing comprises: ethyl cinnamate at 100% concentration.
In some embodiments, the step of tissue clearing comprises: adding the reagent for tissue transparentization into the tissue sample after dehydration treatment, and placing the tissue sample in a shaking table for tissue transparentization treatment.
In some embodiments, the rotational speed of the shaker is from 40 to 60 rpm; the temperature is as follows: 35-40 ℃; preferably, the rotational speed of the shaker is 50 revolutions per minute; the temperature is as follows: 37 ℃ is carried out.
In some embodiments, the tissue clearing treatment is for a time period of: 20 to 30h; preferably, the time of the tissue transparentizing treatment is: and (5) 24h.
In some embodiments, the ratio of the amount of reagent added to the tissue clearing to the volume of the tissue sample is 20:1.
in summary, the present application includes at least one of the following beneficial technical effects:
(1) The transparentizing method provided by the invention realizes the rapid and complete transparentizing treatment of the large-volume sample of the biological tissue and overcomes the defects in other oily reagent transparentizing technologies.
(2) The degreasing agent adopts ethers as a main degreasing agent, and improves the degreasing performance of the degreasing agent by compounding with the surfactant, such as reducing the critical micelle concentration, increasing the wettability and the solubilization, improving the emulsifying capacity and the washing capacity, realizing the degreasing process of a biological tissue sample, and further realizing the transparency of the tissue.
(3) The transparentizing method provided by the invention improves the problems of heavy autofluorescence of the tissue sample and large optical imaging background noise, and the degreasing agent used by the method is low in use concentration, clean and environment-friendly.
Drawings
FIG. 1 shows the results of the transparentization of the mouse brain intact tissue by means of the inventive example;
FIG. 2 shows the result of microscope imaging after the whole mouse brain tissue is subjected to a transparentization treatment by way of an embodiment of the present invention;
FIG. 3 shows the results of the brain clearing of the mouse in comparative example 1, in which the concentration of ethylenediamine was 50%;
FIG. 4 shows the critical micelle concentrations corresponding to the mixed surfactants obtained by different compounding ratios of sodium alcohol ether sulfate and alcohol ether in comparative example 2;
FIG. 5 shows the results of transparentization of mouse brain at 20% concentration after compounding sodium alcohol ether sulfate with alcohol ether in comparative example 3;
FIG. 6 is the result of the mouse brain clearing when absolute ethyl alcohol was used alone as the dehydrating reagent for the S4 solution in comparative example 4.
Detailed Description
The technical solutions of the present invention are further illustrated by the following specific examples, which do not represent limitations to the scope of the present invention. Insubstantial modifications and adaptations of the present invention by others based on the teachings of the present invention are within the scope of the invention.
The devices, materials, and the like used in the following examples are commercially available or publicly available, unless otherwise specified.
EXAMPLE 1 method for transparentizing large-volume sample of biological tissue
Firstly, injecting aorta after mouse anesthesia, taking the whole mouse brain, and placing the whole mouse brain in a 16% paraformaldehyde solution for fixation for 24 hours;
step two, taking out the fixed brain tissue, placing the brain tissue in a test tube, adding 20 ml of S1 solution (10% w/v ethylenediamine, 0.1% w/v sodium hydroxide and pure water as a solvent) into the test tube, placing the test tube in a shaking table, setting the rotation speed to be 50 revolutions per minute and the temperature to be 37 ℃, and starting shaking, heating and incubating for 24 hours;
pouring out the S1 solution, adding 20 ml of the S2 solution (1% v/v fatty alcohol-polyoxyethylene ether and 1% v/v fatty alcohol-polyoxyethylene ether sodium sulfate) into the test tube, placing the test tube in a shaking table, setting the rotation speed to be 50 revolutions per minute and the temperature to be 37 ℃, and starting shaking, heating and incubating for 24 hours;
pouring out the S2 solution, adding 20 ml of the S2 solution (1% v/v fatty alcohol-polyoxyethylene ether and 1% v/v fatty alcohol-polyoxyethylene ether sodium sulfate) into the test tube, placing the test tube in a shaking table, setting the rotation speed to be 50 revolutions per minute and the temperature to be 37 ℃, and starting shaking, heating and incubating for 24 hours;
pouring out the S2 solution, adding 20 ml of S3 solutions with different concentration gradients (the concentration gradients of the added S3 solutions are 50% v/v of ethanol and the balance of pure water v/v in sequence, soaking for 4h, 70% v/v of ethanol and the balance of pure water v/v, soaking for 4h, 90% v/v of ethanol and the balance of pure water v/v, soaking for 4 h) into a test tube, placing the test tube into a shaking table, setting the rotation speed to be 50 revolutions per minute, setting the temperature to be 37 ℃, and starting shaking and heating for incubation for 12 hours;
pouring out the S3 solution, adding 20 ml of the S4 solution (40% v/v of absolute ethyl alcohol and 60% v/v of xylene) into the test tube, placing the test tube in a shaking table, setting the rotation speed to be 50 revolutions per minute and the temperature to be 37 ℃, and starting shaking, heating and incubating for 24 hours;
and seventhly, pouring out the S4 solution, adding 20 ml of the S5 solution (ethyl cinnamate, the concentration of the ethyl cinnamate is 100%) into the test tube, placing the test tube into a shaking table, setting the rotation speed to be 50 r/min and the temperature to be 37 ℃, and starting shaking, heating and incubating for 24 hours until the tissues are completely transparent. The whole mouse brain after the transparentization treatment is obtained, the specific result is shown in fig. 1, and fig. 1 is a top view of the whole mouse brain after the transparentization treatment. The results in FIG. 1 show that: the transparency of the visible rat brain is good, and the characters below the visible rat brain are clear and distinguishable, so that the requirement of performing optical imaging by using a microscope is met.
Example 2 method for transparentizing large-volume biological tissue sample
Firstly, perfusing an aorta after a mouse is anesthetized, and placing a complete mouse brain in a 16% paraformaldehyde solution for fixing for 24 hours;
step two, taking out the fixed brain tissue, placing the brain tissue into a test tube, adding 20 ml of S1 solution (20% w/v ethylenediamine, 5% w/v sodium hydroxide and pure water as solvent) into the test tube, placing the test tube into a shaking table, setting the rotation speed to be 50 revolutions per minute and the temperature to be 37 ℃, and starting shaking, heating and incubating for 24 hours;
pouring out the S1 solution, adding 20 ml of the S2 solution (fatty alcohol-polyoxyethylene ether 1% v/v, fatty alcohol-polyoxyethylene ether sodium sulfate 5% v/v) into the test tube, placing the test tube in a shaking table, setting the rotation speed to be 50 revolutions per minute and the temperature to be 37 ℃, and starting shaking, heating and incubating for 24 hours;
pouring out the S2 solution, adding 20 ml of the S2 solution (fatty alcohol-polyoxyethylene ether 1% v/v, fatty alcohol-polyoxyethylene ether sodium sulfate 5% v/v) into the test tube, placing the test tube in a shaking table, setting the rotation speed to be 50 revolutions per minute and the temperature to be 37 ℃, and starting shaking, heating and incubating for 24 hours;
pouring out the S2 solution, adding 20 ml of S3 solutions with different concentration gradients (the concentration gradients of the added S3 solutions are 50% v/v of ethanol and the balance of pure water v/v in sequence, soaking for 4h, 70% v/v of ethanol and the balance of pure water v/v, soaking for 4h, 90% v/v of ethanol and the balance of pure water v/v, soaking for 4 h) into a test tube, placing the test tube into a shaking table, setting the rotation speed to be 50 revolutions per minute, setting the temperature to be 37 ℃, and starting shaking and heating for incubation for 12 hours;
pouring out the S3 solution, adding 20 ml of the S4 solution (50% v/v of absolute ethyl alcohol and 50% v/v of dimethylbenzene) into the test tube, placing the test tube in a shaking table, setting the rotation speed to be 50 revolutions per minute and the temperature to be 37 ℃, and starting shaking, heating and incubating for 24 hours;
and seventhly, pouring out the S4 solution, adding 20 ml of the S5 solution (ethyl cinnamate, wherein the concentration of the ethyl cinnamate is 100%) into the test tube, placing the test tube into a shaking table, setting the rotation speed at 50 r/min and the temperature at 37 ℃, starting shaking, heating and incubating for 24 hours until the tissue is completely transparent, and thus obtaining the transparent complete mouse brain.
EXAMPLE 3 method for transparentizing large-volume biological tissue sample
Firstly, perfusing an aorta after a mouse is anesthetized, and placing a complete mouse brain in a 16% paraformaldehyde solution for fixing for 24 hours;
step two, taking out the fixed brain tissue, placing the brain tissue into a test tube, adding 20 ml of S1 solution (30% w/v ethylenediamine, 1% w/v sodium hydroxide and pure water as solvent) into the test tube, placing the test tube into a shaking table, setting the rotation speed to be 50 revolutions per minute and the temperature to be 37 ℃, and starting shaking, heating and incubating for 24 hours;
pouring out the S1 solution, adding 20 ml of the S2 solution (5% v/v fatty alcohol-polyoxyethylene ether and 1% v/v fatty alcohol-polyoxyethylene ether sodium sulfate) into a test tube, placing the test tube in a shaking table, setting the rotation speed to be 50 revolutions per minute and the temperature to be 37 ℃, and starting shaking, heating and incubating for 24 hours;
pouring out the S2 solution, adding 20 ml of the S2 solution (5% v/v fatty alcohol-polyoxyethylene ether and 1% v/v fatty alcohol-polyoxyethylene ether sodium sulfate) into the test tube, placing the test tube in a shaking table, setting the rotation speed to be 50 revolutions per minute and the temperature to be 37 ℃, and starting shaking, heating and incubating for 24 hours;
pouring out the S2 solution, adding 20 ml of S3 solutions with different concentration gradients (the concentration gradients of the added S3 solutions are 50% v/v of ethanol and the balance of pure water v/v in sequence, soaking for 4h, 70% v/v of ethanol and the balance of pure water v/v, soaking for 4h, 90% v/v of ethanol and the balance of pure water v/v, soaking for 4 h) into a test tube, placing the test tube into a shaking table, setting the rotation speed to be 50 revolutions per minute, setting the temperature to be 37 ℃, and starting shaking and heating for incubation for 12 hours;
pouring out the S3 solution, adding 20 ml of the S4 solution (60% v/v of absolute ethanol and 40% v/v of xylene) into the test tube, placing the test tube in a shaking table, setting the rotation speed to be 50 r/min and the temperature to be 37 ℃, and starting shaking, heating and incubating for 24 hours;
and seventhly, pouring out the S4 solution, adding 20 ml of the S5 solution (ethyl cinnamate, wherein the concentration of the ethyl cinnamate is 100%) into the test tube, placing the test tube into a shaking table, setting the rotation speed to be 50 r/min and the temperature to be 37 ℃, starting shaking, heating and incubating for 24 hours until the tissue is completely transparent, and thus obtaining the transparent complete mouse brain.
The formulations of the reagents used in examples 1-3 are shown in Table 1:
TABLE 1 formulation of reagents used in examples 1-3
Imaging and observing the whole mouse brain after the transparentization treatment of the embodiment, injecting GFP-labeled virus into a certain nucleus in the mouse brain, dissecting after 3 weeks, taking out the brain, transparentizing the mouse brain by the method, scanning and imaging by using a light sheet microscope, and performing three-dimensional reconstruction on the image to obtain an image, wherein nerve fibers of the virus projected from the injected nucleus to a downstream nucleus can be seen in the image. Specifically, as shown in fig. 2, fig. 2 is an image obtained in example 2, GFP-labeled virus is injected into a certain nucleus in the brain of a mouse, after 3 weeks, the brain is dissected and removed, the mouse brain is cleared by the method of example 2, then scanned and imaged by a light sheet microscope, and then three-dimensional reconstruction of the image is performed to obtain an image shown in fig. 2, which shows that the virus projects from the injected nucleus to nerve fibers of a downstream nucleus.
Comparative example 1
This comparative example is based on example 2 and differs from example 2 in that: the amount of ethylenediamine in the S1 solution used in the first step is different from that in the example 2, and the other conditions are the same as those in the step 2, specifically as follows:
firstly, injecting aorta after mouse anesthesia, taking the whole mouse brain, and placing the whole mouse brain in a 16% paraformaldehyde solution for fixation for 24 hours;
step two, taking out the fixed brain tissue, placing the brain tissue in a test tube, adding 20 ml of S1 solution (50% w/v ethylenediamine, 5% w/v sodium hydroxide and pure water as a solvent) into the test tube, placing the test tube in a shaking table, setting the rotation speed to be 50 revolutions per minute and the temperature to be 37 ℃, and starting shaking, heating and incubating for 24 hours;
pouring out the S1 solution, adding 20 ml of the S2 solution (fatty alcohol-polyoxyethylene ether 1% v/v, fatty alcohol-polyoxyethylene ether sodium sulfate 5% v/v) into the test tube, placing the test tube in a shaking table, setting the rotation speed to be 50 revolutions per minute and the temperature to be 37 ℃, and starting shaking, heating and incubating for 24 hours;
pouring out the S2 solution, adding 20 ml of the S2 solution (fatty alcohol-polyoxyethylene ether 1% v/v, fatty alcohol-polyoxyethylene ether sodium sulfate 5% v/v) into the test tube, placing the test tube in a shaking table, setting the rotation speed to be 50 revolutions per minute and the temperature to be 37 ℃, and starting shaking, heating and incubating for 24 hours;
pouring out the S2 solution, adding 20 ml of S3 solutions with different concentration gradients (the concentration gradients of the added S3 solutions are 50% v/v of ethanol and the balance of pure water v/v in sequence, soaking for 4h, 70% v/v of ethanol and the balance of pure water v/v, soaking for 4h, 90% v/v of ethanol and the balance of pure water v/v, soaking for 4 h) into a test tube, placing the test tube into a shaking table, setting the rotation speed to be 50 revolutions per minute, setting the temperature to be 37 ℃, and starting shaking and heating for incubation for 6-12 hours;
pouring out the S3 solution, adding 20 ml of the S4 solution (50% v/v of absolute ethanol and 50% v/v of xylene) into the test tube, placing the test tube in a shaking table, setting the rotation speed to be 50 r/min and the temperature to be 37 ℃, and starting shaking, heating and incubating for 24 hours;
and seventhly, pouring out the S4 solution, adding 20 ml of the S5 solution (ethyl cinnamate, wherein the concentration of the ethyl cinnamate is 100%) into the test tube, placing the test tube into a shaking table, setting the rotation speed at 50 r/min and the temperature at 37 ℃, starting shaking, heating and incubating for 24 hours until the tissue is completely transparent, and thus obtaining the transparent complete mouse brain.
The obtained whole mouse brain tissue after the transparentization treatment is shown in fig. 3, and the results show that: when the concentration of ethylenediamine in the decolorizing agent S1 solution is 50%, the tissue decolorizing effect is poor. When the concentration of ethylenediamine in the decolorizing reagent S1 solution is 50%, tissues tend to syneresis, and the permeability of ethylenediamine is poor, so that the ethylenediamine is difficult to reach deep parts of the tissues, and the decolorizing effect inside the tissues is influenced; in the technical scheme of the invention, the effect of the degreasing part depends on the expansion and loosening effect of tissues by ethylenediamine.
In the process of exploration, it is found that when the concentration of ethylenediamine in the solution of the decolorizing agent S1 is less than 10%, although the decolorizing effect can be achieved, the decolorizing time is obviously prolonged to more than 48 hours, and the efficiency of the transparentization treatment is further influenced.
Comparative example 2
The comparative example explores the compounding ratio of the sodium alcohol ether sulphate and the fatty alcohol-polyoxyethylene ether in the degreasing reagent of the S2 solution, and the sodium alcohol ether sulphate and the fatty alcohol-polyoxyethylene ether in the degreasing reagent of the S2 solution are compounded according to different proportions, as shown in Table 2:
TABLE 2 compounding ratio Table of sodium fatty alcohol polyoxyethylene ether sulfate and fatty alcohol polyoxyethylene ether
| Serial number | Sodium fatty alcohol polyoxyethylene ether sulfate/fatty |
| 1 | 100 |
| 2 | 50 |
| 3 | 25 |
| 4 | 10 |
| 5 | 5 |
| 6 | 1 |
| 7 | 0.1 |
Further investigating the critical micelle concentration (cmc) of the mixed surfactant obtained by different compounding ratios of the sodium alcohol ether sulphate and the alcohol ether, the result is shown in fig. 4, and the result in fig. 4 shows that: when the ratio of the sodium fatty alcohol-polyoxyethylene ether sulfate to the fatty alcohol-polyoxyethylene ether is close to 5, the critical micelle concentration (cmc) is minimum, the degreasing capability of the obtained surfactant is strongest, and the time required for degreasing is shortest.
Comparative example 3
This comparative example is based on example 2 and differs from example 2 in that: the concentration of the sodium alcohol ether sulphate and the fatty alcohol-polyoxyethylene ether in the S2 solution degreasing reagent used after compounding is different from that in example 2, the concentration of the sodium alcohol ether sulphate and the fatty alcohol-polyoxyethylene ether in the S2 solution degreasing reagent in the comparative example after compounding is 20% (corresponding to 10% v/v of fatty alcohol-polyoxyethylene ether and 10% v/v of fatty alcohol-polyoxyethylene ether sodium sulphate), the rest conditions are the same as those in example 2, the whole mouse brain is subjected to the transparentization treatment in the manner of example 2, and the obtained transparentization treatment result is shown in FIG. 5.
The results in FIG. 5 show that: when the concentration of the compounded surfactant is too high, such as 20%, the volume of the tissue is reduced, the degreasing speed is slow, and complete degreasing cannot be achieved. In the course of the research, it was also found that when the concentration of the compounded surfactant is too low, for example, less than 0.1%, the degreasing time is prolonged, and the complete degreasing effect cannot be achieved.
Comparative example 4
This comparative example is based on example 2 and differs from example 2 in that: the dehydrating reagent of the S4 solution used is different from that of the example 2, and the dehydrating reagents of the S4 solution in the comparative example are respectively: the whole mouse brain was cleared using absolute ethanol alone or xylene alone under the same conditions as in example 2, and the clearing results are shown in FIG. 6, in the same manner as in example 2.
The results in FIG. 6 show that: when absolute ethyl alcohol is used as the S4 solution dehydration reagent alone, tissues cannot be transparent. In the course of the research, it was found that when xylene alone was used as the dehydrating reagent for the S4 solution, the tissue could not be transparent as well as when absolute ethanol alone was used, and no good effect of transparentization could be achieved.
It is to be understood that the present invention has been described with reference to certain embodiments, and that various changes in the features and embodiments, or equivalent substitutions may be made therein by those skilled in the art without departing from the spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from the essential scope thereof. Therefore, it is intended that the invention not be limited to the particular embodiment disclosed, but that the invention will include all embodiments falling within the scope of the appended claims.
Claims (10)
1. A method for transparentizing a large-volume sample of biological tissue, which is characterized by comprising the following steps:
(1) Fixing;
(2) Decoloring;
(3) Degreasing;
(4) Dehydrating;
(5) Clearing the tissue;
wherein in the step (3), the reagent used for degreasing is: compounding polyether substances and salt to obtain the polyether compound; the compounding ratio of the polyether substance to the salt is as follows in terms of v/v: 1:0.2 to 10 parts by weight.
2. The method of claim 1, wherein the reagent used for degreasing has a concentration of 0.2% to 15% in v/v.
3. The method of claim 1, wherein the reagents used for degreasing comprise, in v/v: 0.1 to 9 percent of polyether substance and 0.1 to 9 percent of salt.
4. The method of claim 1, wherein the polyether is selected from one or more of fatty alcohol polyoxyethylene ether, alkylphenol polyoxyethylene ether, castor oil polyoxyethylene ether and lauric acid polyoxyethylene ether; the salt is selected from one or more of fatty alcohol-polyoxyethylene ether sodium sulfate, lauryl sodium sulfate and triethanolamine.
5. The method of claim 1, wherein in step (3), the step of degreasing comprises: adding a reagent for degreasing into the decolored tissue sample, and placing the tissue sample in a shaking table for degreasing; wherein the degreasing times are as follows: is more than or equal to 1 time.
6. The method according to claim 1, wherein in the step (2), the reagents for decolorization comprise, in w/v: 1 to 40 percent of ethylenediamine, 0.1 to 10 percent of sodium hydroxide and the balance of purified water.
7. The method of claim 1, wherein in step (4), the dehydrated reagent comprises a first dehydrated reagent, a second dehydrated reagent; the first dehydrating reagent comprises, in v/v: ethanol water solution with different concentration gradients; the second dehydrating reagent comprises, in v/v: absolute ethanol and xylene.
8. The method of claim 7, wherein the first dehydrating reagent comprises, in v/v: 40-60% ethanol water solution, 60-80% ethanol water solution and 80-95% ethanol water solution; the second dehydrating reagent comprises: 10 to 90 percent of absolute ethyl alcohol and 90 to 10 percent of dimethylbenzene.
9. The method of claim 7, wherein the step of dewatering comprises:
1) Sequentially adding the first dehydration reagent into the tissue sample after the degreasing treatment, and placing the tissue sample in a shaking table for primary dehydration treatment;
2) Adding the second dehydration reagent into the tissue sample subjected to the primary dehydration treatment in the step 1), and placing the tissue sample in a shaking table for secondary dehydration treatment.
10. The method of claim 1, wherein in step (5), the agent for tissue clearing is ethyl cinnamate at a concentration of 100%.
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