CN115791339B - Transparentization method of biological tissue large-volume sample - Google Patents
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Abstract
The invention belongs to the technical field of biological tissue transparentization, and relates to a transparentization method of a large-volume sample of biological tissue, which comprises the following steps: (1) fixing; (2) decoloring; (3) degreasing; (4) dehydration; (5) tissue transparentization; wherein in the step (3), the degreasing agent is: and (3) 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 in 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 of a large-volume sample of biological tissue.
Background
With the continuous development of biomedicine, three-dimensional reconstruction of tissue structures plays an increasingly important role in histomorphology and physiological and pathological research. Common medical imaging techniques such as Computed Tomography (CT) and Magnetic Resonance Imaging (MRI) reveal three-dimensional anatomy of living beings, but it is difficult to obtain tissue structures at the cellular resolution level. Modern optical slice microscopic imaging technologies such as confocal and two-photon microscopes can realize three-dimensional imaging of tissues without physical slicing, and imaging depth can reach tens to hundreds of micrometers. However, complete imaging of large volumes of tissue remains a challenge due to the large number of thick tissue layers, non-uniform refractive index of the internal material. The biological tissue bulk sample is opaque, if the tissue cell structure in the biological tissue bulk sample is required to be observed, the biological tissue bulk sample is cut into slices, such as paraffin sections, frozen sections and the like, and the tissue slices are placed in a microscopic optical system for imaging observation.
Tissue transparentization technology which is emerging in recent years is a method of soaking by chemical reagents to make tissues transparent, and complete three-dimensional imaging of the tissues is realized by means of emerging optical imaging tools such as a light sheet microscope without cutting the tissues. Various tissue transparentization techniques are rapidly developed, and the current stage transparentization techniques are mainly divided into 3 types: hydrophilic tissue transparentization technology, hydrophobic tissue transparentization technology, and hydrogel embedding method. Correspondingly, the transparentizing agent is divided into: water-soluble reagent, oily reagent, hydrogel reagent.
Research literature proves that the degreasing agent is one of the causes of the primary immunofluorescence loss of the tissue sample, and most of the methods disclosed in the prior art adopt alcohols and esters as the degreasing agent, so that the problems of heavy autofluorescence, large optical imaging background noise and the like of the tissue sample after the transparentization treatment are caused by the immunofluorescence loss.
Disclosure of Invention
The invention aims to provide a method for transparentizing a large-volume sample of biological tissues.
In one aspect, the invention provides a method for transparentizing a large-volume sample of biological tissue, comprising the steps of:
(1) Fixing;
(2) Decoloring;
(3) Degreasing;
(4) Dehydrating;
(5) Transparentizing the tissue;
wherein in the step (3), the degreasing agent is: the polyether substance and the corresponding salt are compounded to obtain the polyether compound; the compound proportion of the polyether substance and the salt is as follows in terms of v/v: 1: 0.2-10.
Research literature proves that the degreasing agent is one of the causes of the primary immunofluorescence loss of the tissue sample, and most of the methods disclosed in the prior art adopt alcohols and esters as the degreasing agent, so that the problems of heavy autofluorescence, large optical imaging background noise and the like of the tissue sample after the transparentization treatment are caused by the immunofluorescence loss. The invention adopts ethers as degreasing agent and is compounded with the surfactant, thereby improving the problems of primary immunofluorescence loss of the tissue sample, heavy autofluorescence of the tissue sample after the transparentization treatment, large optical imaging background noise and the like.
In some embodiments, the polyether substance and salt are compounded in a ratio of v/v of: 1: 0.2-6; preferably, the polyether substance and the salt are compounded according to the ratio of v/v: 1: 4-6.
In some embodiments, the concentration of the degreasing agent is 0.2% -15% in v/v; preferably, the concentration of the agent used for degreasing is 1% -10%.
In some embodiments, the reagents used for degreasing in v/v include: 0.1 to 9 percent of polyether substance and 0.1 to 9 percent of salt; preferably, the agent for degreasing comprises: 1 to 5 percent of polyether substance and 1 to 5 percent of salt; preferably, the agent for degreasing comprises: 1 to 5 percent of polyether substance and 1 to 5 percent of salt; preferably, the agent for degreasing comprises: 1% of polyether substance and 5% of salt.
In some embodiments, the polyether substance is selected from any one or more of fatty alcohol polyoxyethylene ether, alkylphenol polyoxyethylene ether, castor oil polyoxyethylene ether, lauric acid polyoxyethylene ether.
In some embodiments, the salt is selected from any one or more of sodium fatty alcohol polyoxyethylene ether sulfate, sodium dodecyl sulfate, triethanolamine.
In some embodiments, in step (3), the degreasing step comprises: adding a degreasing reagent into the decolorized tissue sample, and placing the tissue sample in a shaking table for degreasing; wherein, the times of degreasing are as follows: and 1 or more times.
In some embodiments, the number of degreasing is: 1-2 times; preferably, the number of degreasing times is: 2 times.
In some embodiments, the rotation speed of the shaking table is 40-60 revolutions per minute; the temperature is as follows: 35-40 ℃; preferably, the rotation speed of the cradle is 50 rpm; the temperature is as follows: 37 ℃.
In some embodiments, the degreasing time is: 20-30 h; preferably, the degreasing time is as follows: 24h.
In some embodiments, the degreasing agent is added in an amount to volume ratio of 20:1.
in some embodiments, in step (1), the reagent used for the immobilization is: 15-20% of paraformaldehyde solution; preferably, the fixed time is: 12-24 hours.
In some embodiments, in step (2), the agent used for the decolorization comprises, 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 agent used for decoloring comprises: 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 placing the tissue sample in a shaking table for decoloring; preferably, the rotating speed of the shaking table is 40-60 revolutions per minute; the temperature is as follows: 35-40 ℃; preferably, the decolorizing time is: 20-30 h; preferably, the ratio of the addition of the reagent used 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 dehydrating reagent comprises, in v/v: ethanol water solutions with different concentration gradients; the second dehydrating reagent comprises, in v/v: absolute ethanol and xylene.
In some embodiments, the first dehydrating 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 dehydrating reagent comprises, in v/v: 10-90% of absolute ethyl alcohol and 90-10% of dimethylbenzene; preferably, the second dehydrating reagent comprises: 40-60% of absolute ethyl alcohol and 60-40% of dimethylbenzene.
In some embodiments, the step of dewatering comprises:
1) Sequentially adding the first dehydration reagent into the degreased tissue sample, and placing the tissue sample in a shaking table for one-time dehydration treatment;
2) And (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 to carry out secondary dehydration treatment.
In some embodiments, in the primary dehydration treatment, the rotation speed of the shaking table is 40-60 rpm; the temperature is as follows: 35-40 ℃; preferably, the rotation speed of the cradle is 50 rpm; the temperature is as follows: 37 ℃.
In some embodiments, the time for dehydration is: 6-15 h; preferably, the time for dehydration is: 6-12 h;
in some embodiments, the first decolorizing reagent is added in an amount to volume ratio of 20 to the tissue sample: 1.
in some embodiments, the dehydration time of the ethanol aqueous solutions with different concentration gradients in the first dehydration reagent is 2-5 h respectively; preferably, the dehydration time of the ethanol aqueous solutions with different concentration gradients in the first dehydration reagent is 2-4 hours respectively.
In some embodiments, in the secondary dehydration treatment, the rotation speed of the shaking table is 40-60 revolutions per minute; the temperature is as follows: 35-40 ℃; preferably, the rotation speed of the cradle is 50 rpm; the temperature is as follows: 37 ℃.
In some embodiments, the time for dehydration is: 20-30 h; preferably, the time for dehydration is: 24h.
In some embodiments, the first decolorizing reagent is added in an amount to volume ratio of 20 to the tissue sample: 1.
in some embodiments, in step (5), the agent used for tissue transparentization comprises: ethyl cinnamate at a concentration of 100%.
In some embodiments, the step of tissue transparentizing comprises: and adding the reagent for tissue transparentizing into the dehydrated tissue sample, and placing the tissue sample in a shaking table for tissue transparentizing.
In some embodiments, the rotation speed of the shaking table is 40-60 revolutions per minute; the temperature is as follows: 35-40 ℃; preferably, the rotation speed of the cradle is 50 rpm; the temperature is as follows: 37 ℃.
In some embodiments, the tissue transparentization process is performed for a time of: 20-30 h; preferably, the tissue transparentizing treatment is performed for a time of: 24h.
In some embodiments, the reagent is added in an amount to volume ratio of 20 to the tissue sample for tissue transparency: 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 invention adopts ethers as main degreasing agent, and by compounding with the surfactant, the degreasing performance of the degreasing agent is improved, such as the critical micelle concentration is reduced, the wettability and the solubilisation are increased, the emulsifying capacity and the washing capacity are improved, the degreasing process of biological tissue samples is realized, and the transparency of tissues is further realized.
(3) The transparentizing method provided by the invention solves the problems of heavy autofluorescence and large optical imaging background noise of the tissue sample, and the degreasing agent used by the method is low in use concentration, clean and environment-friendly.
Drawings
FIG. 1 shows the results of transparentizing the whole tissue of the brain of a mouse by means of an inventive example;
FIG. 2 is a microscopic image of the whole tissue of the mouse brain after transparentization by way of example of the invention;
FIG. 3 shows the brain transparentization results of the mice at an ethylenediamine concentration of 50% in comparative example 1;
FIG. 4 shows the critical micelle concentrations of the mixed surfactant obtained by performing different compounding examples on the fatty alcohol-polyoxyethylene ether sodium sulfate and the fatty alcohol-polyoxyethylene ether in comparative example 2;
FIG. 5 shows the result of transparentization of the mouse brain at a concentration of 20% after the combination of the sodium fatty alcohol-polyoxyethylene ether sulfate and the fatty alcohol-polyoxyethylene ether in comparative example 3;
FIG. 6 shows the results of brain transparentization in mice in comparative example 4 using absolute ethanol alone as the dehydration reagent for the S4 solution.
Detailed Description
The technical solution of the present invention is further illustrated by the following specific examples, which do not represent limitations on the scope of the present invention. Some insubstantial modifications and adaptations of the invention based on the inventive concept by others remain within the scope of the invention.
The equipment, materials, and the like used in the examples described below are commercially or publicly available unless otherwise specified.
Example 1A method for transparentizing a large volume sample of biological tissue
Step one, injecting aorta after anesthesia of a mouse, and taking the whole mouse brain to be placed in a 16% paraformaldehyde solution for fixation 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 (ethylenediamine 10% w/v, sodium hydroxide 0.1% w/v, and pure water as solvent) into the test tube, placing the test tube into a shaking table, setting the rotation speed to 50 revolutions per minute, setting the temperature to 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 of fatty alcohol polyoxyethylene ether and 1% v/v of fatty alcohol polyoxyethylene ether sodium sulfate) into a test tube, placing the test tube into a shaking table, setting the rotating speed to be 50 revolutions per minute, setting the temperature to be 37 ℃, and starting shaking, heating and incubating for 24 hours;
pouring out the S2 solution, re-adding 20 ml of the S2 solution (1% v/v of fatty alcohol polyoxyethylene ether and 1% v/v of fatty alcohol polyoxyethylene ether sodium sulfate) into the test tube, placing the test tube into a shaking table, setting the rotating speed to be 50 revolutions per minute, setting the temperature to be 37 ℃, and starting shaking, heating and incubating for 24 hours;
pouring out the S2 solution, adding 20 ml of different concentration gradient S3 solutions into a test tube (the concentration gradient sequence of the S3 solution added here is 50% v/v of ethanol, the balance of pure water v/v, soaking for 4 hours, 70% v/v of ethanol, the balance of pure water v/v, soaking for 4 hours, 90% v/v of ethanol, the balance of pure water v/v, soaking for 4 hours), placing the test tube into a shaking table, setting the rotating speed to be 50 revolutions per minute, setting the temperature to be 37 ℃, and starting shaking, heating and incubating 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 dimethylbenzene) into a test tube, placing the test tube in a shaking table, setting the rotating speed to be 50 revolutions per minute, setting the temperature to be 37 ℃, and starting shaking, heating and incubating for 24 hours;
pouring out the S4 solution, adding 20 ml of the S5 solution (ethyl cinnamate with the concentration of 100%) into the test tube, placing the test tube into a shaking table, setting the rotating speed to be 50 revolutions per minute, setting the temperature to be 37 ℃, starting shaking and heating for incubation for 24 hours, and finally, completely transparentizing the tissue. The whole brain of the mice after the transparentization is obtained, the specific result is shown in figure 1, and figure 1 is a top view of the whole brain of the mice after the transparentization. The results in fig. 1 show that: the visible mouse brain has good transparency, and the lower font is clear and distinguishable, so that the requirement of microscopic optical imaging can be met.
Example 2A method for transparentizing a large volume sample of biological tissue
Step one, injecting aorta after anesthesia of a mouse, and taking the whole mouse brain to be placed in a 16% paraformaldehyde solution for fixation 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 (ethylenediamine 20% w/v, sodium hydroxide 5% w/v, and pure water as solvent) into the test tube, placing the test tube into a shaking table, setting the rotation speed to 50 rpm, setting the temperature to 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 of fatty alcohol polyoxyethylene ether and 5% v/v of fatty alcohol polyoxyethylene ether sodium sulfate) into a test tube, placing the test tube into a shaking table, setting the rotating speed to be 50 revolutions per minute, setting the temperature to be 37 ℃, and starting shaking, heating and incubating for 24 hours;
pouring out the S2 solution, re-adding 20 ml of the S2 solution (1% v/v of fatty alcohol polyoxyethylene ether and 5% v/v of fatty alcohol polyoxyethylene ether sodium sulfate) into the test tube, placing the test tube into a shaking table, setting the rotating speed to be 50 revolutions per minute, setting the temperature to be 37 ℃, and starting shaking, heating and incubating for 24 hours;
pouring out the S2 solution, adding 20 ml of different concentration gradient S3 solutions into a test tube (the concentration gradient sequence of the S3 solution added here is 50% v/v of ethanol, the balance of pure water v/v, soaking for 4 hours, 70% v/v of ethanol, the balance of pure water v/v, soaking for 4 hours, 90% v/v of ethanol, the balance of pure water v/v, soaking for 4 hours), placing the test tube into a shaking table, setting the rotating speed to be 50 revolutions per minute, setting the temperature to be 37 ℃, and starting shaking, heating and incubating 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 a test tube, placing the test tube in a shaking table, setting the rotating speed to be 50 revolutions per minute, setting the temperature to be 37 ℃, and starting shaking, heating and incubating for 24 hours;
pouring out the S4 solution, adding 20 ml of the S5 solution (ethyl cinnamate with the concentration of 100%) into a test tube, placing the test tube into a shaking table, setting the rotating speed to be 50 revolutions per minute, setting the temperature to be 37 ℃, starting shaking, heating and incubating for 24 hours until the tissue is completely transparent, and obtaining the whole mouse brain after the transparentization treatment.
Example 3A method for transparentizing a large volume sample of biological tissue
Step one, injecting aorta after anesthesia of a mouse, and taking the whole mouse brain to be placed in a 16% paraformaldehyde solution for fixation 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 (ethylenediamine 30% w/v, sodium hydroxide 1% w/v, and pure water as solvent) into the test tube, placing the test tube into a shaking table, setting the rotation speed to 50 revolutions per minute, setting the temperature to 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 of fatty alcohol polyoxyethylene ether and 1% v/v of fatty alcohol polyoxyethylene ether sodium sulfate) into a test tube, placing the test tube into a shaking table, setting the rotating speed to be 50 revolutions per minute, setting the temperature to be 37 ℃, and starting shaking, heating and incubating for 24 hours;
pouring out the S2 solution, re-adding 20 ml of the S2 solution (5% v/v of fatty alcohol polyoxyethylene ether and 1% v/v of fatty alcohol polyoxyethylene ether sodium sulfate) into the test tube, placing the test tube into a shaking table, setting the rotating speed to be 50 revolutions per minute, setting the temperature to be 37 ℃, and starting shaking, heating and incubating for 24 hours;
pouring out the S2 solution, adding 20 ml of different concentration gradient S3 solutions (the concentration gradient sequence of the added S3 solution is 50% v/v of ethanol, the rest is pure water v/v, soaking for 4 hours, 70% v/v of ethanol, the rest is pure water v/v, soaking for 4 hours, 90% v/v of ethanol, the rest is pure water v/v, soaking for 4 hours), placing the test tube in a shaking table, setting the rotating speed to be 50 revolutions per minute, setting the temperature to be 37 ℃, and starting shaking, heating and incubating for 12 hours;
pouring out the S3 solution, adding 20 ml of the S4 solution (60% v/v of absolute ethyl alcohol and 40% v/v of dimethylbenzene) into a test tube, placing the test tube in a shaking table, setting the rotating speed to be 50 revolutions per minute, setting the temperature to be 37 ℃, and starting shaking, heating and incubating for 24 hours;
pouring out the S4 solution, adding 20 ml of the S5 solution (ethyl cinnamate with the concentration of 100%) into a test tube, placing the test tube into a shaking table, setting the rotating speed to be 50 revolutions per minute, setting the temperature to be 37 ℃, starting shaking, heating and incubating for 24 hours until the tissue is completely transparent, and obtaining the whole mouse brain after the transparentization treatment.
The formulations of the reagents used in examples 1-3 are shown in Table 1:
TABLE 1 formulation of the reagents used in examples 1-3
Imaging and observing the whole mouse brain after the above example is subjected to transparentization, injecting GFP-marked virus into a certain nucleus in the mouse brain, dissecting and taking the brain after 3 weeks, scanning and imaging the mouse brain by using a light sheet microscope after the above method is used for transparentization, and then carrying out three-dimensional image reconstruction to obtain an image, wherein the nerve fibers of the virus projected from the injected nucleus to the downstream nucleus can be seen in the image. Specifically, as shown in fig. 2, fig. 2 is an image obtained in example 2, GFP-tagged virus was injected into a nucleus in the brain of a mouse, after 3 weeks, the brain was dissected and removed, the brain of the mouse was transparentized by the method of example 2, then scanned and imaged by a light sheet microscope, and then three-dimensional image reconstruction was performed to obtain an image as shown in fig. 2, and it was seen that the virus was projected from the injected nucleus to nerve fibers of the 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 example 2, and the other conditions and steps are the same as in example 2, and specifically the following steps are adopted:
step one, injecting aorta after anesthesia of a mouse, and taking the whole mouse brain to be placed in a 16% paraformaldehyde solution for fixation 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 (ethylenediamine 50% w/v, sodium hydroxide 5% w/v, and pure water as solvent) into the test tube, placing the test tube into a shaking table, setting the rotation speed to 50 rpm, setting the temperature to 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 of fatty alcohol polyoxyethylene ether and 5% v/v of fatty alcohol polyoxyethylene ether sodium sulfate) into a test tube, placing the test tube into a shaking table, setting the rotating speed to be 50 revolutions per minute, setting the temperature to be 37 ℃, and starting shaking, heating and incubating for 24 hours;
pouring out the S2 solution, re-adding 20 ml of the S2 solution (1% v/v of fatty alcohol polyoxyethylene ether and 5% v/v of fatty alcohol polyoxyethylene ether sodium sulfate) into the test tube, placing the test tube into a shaking table, setting the rotating speed to be 50 revolutions per minute, setting the temperature to be 37 ℃, and starting shaking, heating and incubating for 24 hours;
pouring out the S2 solution, adding 20 ml of different concentration gradient S3 solutions (the concentration gradient sequence of the added S3 solution is that ethanol is 50% v/v, the rest is pure water v/v, soaking for 4 hours, ethanol is 70% v/v, the rest is pure water v/v, soaking for 4 hours, ethanol is 90% v/v, the rest is pure water v/v, soaking for 4 hours), placing the test tube in a shaking table, setting the rotating speed to be 50 revolutions per minute, setting the temperature to be 37 ℃, and starting shaking and incubating for 6-12 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 a test tube, placing the test tube in a shaking table, setting the rotating speed to be 50 revolutions per minute, setting the temperature to be 37 ℃, and starting shaking, heating and incubating for 24 hours;
pouring out the S4 solution, adding 20 ml of the S5 solution (ethyl cinnamate with the concentration of 100%) into a test tube, placing the test tube into a shaking table, setting the rotating speed to be 50 revolutions per minute, setting the temperature to be 37 ℃, starting shaking, heating and incubating for 24 hours until the tissue is completely transparent, and obtaining the whole mouse brain after the transparentization treatment.
The resulting transparentized whole mouse brain tissue is shown in fig. 3, which shows that: when the ethylenediamine concentration in the decolorization reagent S1 solution is 50%, the tissue is less decolorized. When the concentration of ethylenediamine in the decolorizing agent S1 solution is 50%, the tissue tends to dehydrate and shrink, and the permeability of ethylenediamine is poor, so that ethylenediamine is difficult to reach deep parts of the tissue, and the decolorizing effect inside the tissue is affected; the insufficient tissue expansion can further influence the subsequent degreasing effect, and in the technical scheme of the invention, the degreasing part depends on the expansion loosening effect of the ethylenediamine on the tissue.
In the exploring process, when the concentration of ethylenediamine in the decolorizing agent S1 solution is lower than 10%, the decolorizing effect can be achieved, but the decolorizing time is obviously prolonged to more than 48 hours, and the efficiency of the transparentizing treatment is further affected.
Comparative example 2
The comparative example is a study on the compounding ratio of sodium fatty alcohol-polyoxyethylene ether sulfate and fatty alcohol-polyoxyethylene ether in the S2 solution degreasing reagent, and the compounding ratio of sodium fatty alcohol-polyoxyethylene ether sulfate and fatty alcohol-polyoxyethylene ether in the S2 solution degreasing reagent is different, as shown in table 2:
table 2 table of the compounding ratio of sodium fatty alcohol-polyoxyethylene ether sulfate and fatty alcohol-polyoxyethylene ether
| Sequence number | Sodium fatty alcohol-polyoxyethylene ether sulfate/fatty alcohol- |
| 1 | 100 |
| 2 | 50 |
| 3 | 25 |
| 4 | 10 |
| 5 | 5 |
| 6 | 1 |
| 7 | 0.1 |
Further examining the critical micelle concentration (cmc) of the mixed surfactant obtained by different compounding ratios of the fatty alcohol-polyoxyethylene ether sodium sulfate and the fatty alcohol-polyoxyethylene ether, and the results are shown in the figure 4, and the results in the figure 4 show 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, and the degreasing capability of the obtained surfactant is also strongest, and the degreasing time is also shortest.
Comparative example 3
This comparative example is based on example 2 and differs from example 2 in that: the concentration of the fatty alcohol-polyoxyethylene ether sodium sulfate in the S2 solution degreasing agent after being compounded with the fatty alcohol-polyoxyethylene ether is different from that of the embodiment 2, the concentration of the fatty alcohol-polyoxyethylene ether sodium sulfate in the S2 solution degreasing agent after being compounded with the fatty alcohol-polyoxyethylene ether is 20% (the corresponding fatty alcohol-polyoxyethylene ether is 10% v/v, the fatty alcohol-polyoxyethylene ether sodium sulfate is 10% v/v), the rest conditions are the same as those of the embodiment 2, and the whole brain of the mouse is subjected to the transparentization treatment in the same manner as the embodiment 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, for example, 20%, the shrinkage volume of the tissue is reduced, the degreasing speed is reduced, and complete degreasing cannot be achieved. It was also found during the discovery that when the concentration of the compounded surfactant was too low, e.g., below 0.1%, this resulted in an extended degreasing time and failure to achieve a complete degreasing effect.
Comparative example 4
This comparative example is based on example 2 and differs from example 2 in that: the S4 solution dehydration reagent used was different from example 2 in which the S4 solution dehydration reagents were respectively: the whole brain of the mouse was transparentized in the same manner as in example 2 under the same conditions as in example 2 except that absolute ethanol or xylene was used alone, and the transparentization results are shown in FIG. 6.
The results in fig. 6 show that: when absolute ethyl alcohol is used alone as the dehydration reagent of the S4 solution, the tissue cannot be transparent. In the course of the search, it was found that when xylene alone was used as the dehydration reagent for the S4 solution, the same results as those of absolute ethyl alcohol alone were obtained, and the tissue was not transparent, and the good transparentization effect was not achieved.
It will be understood that the invention has been described in terms of several embodiments, and that various changes and equivalents may be made to these features and embodiments 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 (3)
1. A method for transparentizing a large volume sample of biological tissue, comprising the steps of:
(1) Fixing;
(2) Decoloring;
(3) Degreasing;
(4) Dehydrating;
(5) Transparentizing the tissue;
wherein in the step (3), the degreasing agent is: the polyether substance and the salt are compounded to obtain the compound; the compound proportion of the polyether substance and the salt is as follows in terms of v/v: 1: 0.2-10;
the concentration of the agent for degreasing is 0.2% -15% in terms of v/v;
the reagents used for the 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;
in the step (4), the dehydrated reagent comprises a first dehydrated reagent and a second dehydrated reagent; the first dehydrating reagent comprises, in v/v: ethanol water solutions with different concentration gradients; the second dehydrating reagent comprises, in v/v: absolute ethanol and xylene; the step of dehydrating comprises:
1) Sequentially adding the first dehydration reagent into the degreased tissue sample, and placing the tissue sample in a shaking table for one-time 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 the step (5), the reagent used for tissue transparentization is ethyl cinnamate, and the concentration of the reagent is 100%;
the reagents used for degreasing in v/v include: 0.1 to 9 percent of polyether substance and 0.1 to 9 percent of salt;
the polyether substance 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, sodium dodecyl sulfate and triethanolamine.
2. The method of claim 1, wherein in step (3), the degreasing step comprises: adding a degreasing reagent into the decolorized tissue sample, and placing the tissue sample in a shaking table for degreasing; wherein, the times of degreasing are as follows: and 1 or more times.
3. The method of claim 1, 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 includes: 10-90% of absolute ethyl alcohol and 90-10% of dimethylbenzene.
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