CN113340699A - Organoid tissue frozen section embedding kit and embedding method - Google Patents
Organoid tissue frozen section embedding kit and embedding method Download PDFInfo
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- CN113340699A CN113340699A CN202110611852.4A CN202110611852A CN113340699A CN 113340699 A CN113340699 A CN 113340699A CN 202110611852 A CN202110611852 A CN 202110611852A CN 113340699 A CN113340699 A CN 113340699A
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Abstract
The invention relates to an organoid tissue frozen section embedding kit and an embedding method, which can improve the structural integrity of organoids, reduce the loss of protein antigens, prevent the degradation of genes (RNA and DNA) and greatly improve the preparation success rate of organoid tissue frozen sections.
Description
Technical Field
The invention relates to the technical field of organoids, in particular to a freezing embedding kit and an embedding method for organoids.
Background
Organoids belong to three-dimensional (3D) cell cultures, containing some of the key properties that represent the organ, and are described by Clever et al in Sato T, Vries RG, Snippert HJ, et al, single Lgr5 stem cells builded crypt-viral structures in video with a mesenchyme he, nature.2009may14; 459(7244) 262-5, it was suggested that single cells could form the crypt villus structure of the intestine in isolated culture of small intestine stem cells. In recent years, organoid technology is rapidly developed, and various organoids such as brain, lung, liver, kidney, intestinal tract, stomach and the like are successively established and applied to multiple fields such as precise medical treatment, organ transplantation, drug screening, drug action mechanism research and the like. Organoids have the advantages of being able to simulate signal communication between various cells and the movement of cells in vivo, maintain gene stability during culture, simulate the development of human development and disease, compensate for the deficiencies of animal experiments, and enable real-time imaging.
Cells are dependent on cell adhesion and cytoskeleton for growth in vitro, and since organoids are three-dimensional structures and therefore require support from the corresponding environment, the most common method for inducing organoids to develop three-dimensional properties is to use a solid extracellular matrix that supports cell growth and cell adhesion. At present, the extracellular matrix used mainly includes natural extracellular matrix, Matrigel (also called organism-derived matrix Matrigel), extracellular matrix-chemical hydrogel, and Matrigel is the most widely used one.
The matrigel has the characteristic of presenting different phase states at different temperatures, the matrigel is solid when the temperature is below 20 ℃ below zero, liquid when the temperature is 4 ℃ to 8 ℃, and solidified into jelly shape when the temperature is above 37 ℃, and the culture environment of organoid in the matrigel is a carbon dioxide incubator at 37 ℃, so that the organoid fixed in the matrigel is jelly-shaped, and the special phase state causes that the preparation of frozen slices in the organoid matrigel is very difficult, and the method mainly has the following problems:
firstly, the types of the embedding and fixing reagents of the organoids are more, and the using conditions of various embedding and fixing reagents are different, so that the antigen can not be lost and the gene can not be degraded.
Secondly, the matrigel problem of staining because matrigel itself does not have the colour, consequently can't confirm the position of organoid after fixing with the fixed reagent of embedding, can only blind cut, hardly acquires fine sample position, extravagant experiment consumptive material, can lead to the experiment failure even.
Thirdly, organoid culture well plates are in various styles, from 6-96 wells, organoids can be cultured, the culture wells of different well plates are different in size, and the matrigel is jelly-shaped, so that the matrigel for culturing organoids is difficult to completely take out, and the structure of organoids is easily damaged.
Fourthly, since organoids are basically cultured in cell culture plates, the cell culture plates cannot be directly embedded, and different culture plates have different sizes and numbers of cultured organoids, and no good embedding mold exists.
Therefore, in the preparation of the organoid frozen section sample, the fixation of organoid tissues, the material obtaining of organoid tissues and the organoid tissue embedding mould are all key factors which cause the success of the pre-preparation of the organoid frozen section and are problems to be solved urgently.
Disclosure of Invention
The invention aims to provide an organoid tissue frozen section embedding kit and an embedding method aiming at the defects of the prior art, which can improve the structural integrity of organoids, reduce the loss of protein antigens, prevent the degradation of genes (RNA and DNA) and greatly improve the preparation success rate of organoid tissue frozen sections.
The invention has a technical scheme that: an embedding kit for organ-like tissue frozen sections comprises an outer box, wherein an embedding box, a glue taking device and a group of embedding reagents are arranged in the outer box, the embedding box comprises a barrel body and a bottom plate, the inner diameter of the barrel body is 10-15 mm, the depth of the barrel body is 15-20 mm, the wall thickness of the barrel body is 1-2 mm, an annular positioning groove for the barrel body to insert is formed in the upper end of the bottom plate, the groove width of the annular positioning groove is matched with the wall thickness of the barrel body, an axial through hole is formed in the middle of the bottom plate, a piece of tinfoil paper is fixedly bonded to the lower surface of the bottom plate, the lower end of the axial through hole is sealed through the tinfoil paper, and a barrel cover is arranged at the upper end of the barrel body;
the glue taking device is provided with a handle, one end of the handle is cylindrical, the other end of the handle is flat, the flat end is vertically bent to form a glue taking part, the width of the glue taking part is 3-3.5 mm, the height of the glue taking part is 3-4 mm, and the glue taking part is used for taking out a sample from the culture pore plate and placing the sample on the tin foil paper; the embedding reagent comprises methanol, methylene blue and an OCT embedding agent.
Furthermore, the extension end of the glue taking part is arc-shaped, and the arc top height of the arc is 1 mm.
Further, the length of the handle is 150mm, and the diameter of the handle is the same as the width of the glue taking part.
Further, the barrel cover and the barrel are connected together through a hinge.
Furthermore, the upper surface of the cylinder cover is frosted.
Further, the inner diameter of the cylinder is the same as that of the axial through hole of the bottom plate.
Adopt above-mentioned technical scheme: get the perpendicular bending of flat shape end of gluey ware handle and form and get gluey portion, make and get gluey portion and can insert along the cell culture hole edge of cultivateing the orifice plate, because the matrigel in the cell culture hole is the jelly form, consequently the accessible gets gluey portion and holds up matrigel in the bottom in cell culture hole, and the width of getting gluey portion is 3 ~ 3.5mm, and highly is 3 ~ 4mm, can guarantee to get gluey portion and have certain activity degree of freedom in the cell culture hole, after getting gluey portion and holding matrigel, can hold the handle and make and get gluey portion and rock about, thereby can the biggest whole matrigel of taking out, prevent to destroy the structure of class organ, guarantee the structural integrity of the class organ who takes out. Matrix glue taken out by the glue taking device is placed on tin foil paper of an embedding box, a barrel of the embedding box is inserted into an annular positioning groove of a bottom plate and fixed, so that an organoid is embedded in the barrel, the shape of the barrel is shaped, the whole organoid sample after OCT embedding is conveniently frozen and matched with a bottom support of a freezing microtome, and the lower surface of the bottom plate is fixedly bonded with the tin foil paper, so that the embedded sample can be integrally taken out, the tin foil paper is torn down when the frozen sample is sliced, the sample is lightly pushed from an upper port of the barrel, the sample can be pushed out from an axial through hole of the bottom plate, and the integrity of the organoid structure is ensured. The embedding kit is simple in structure and convenient to use, can ensure the completeness of the organoid structure to the maximum extent, and improves the preparation success rate of the organoid frozen section. And, because embedding reagent and embedding box, get and adorn in an embedding reagent box together with gluey ware, the experimenter just can directly utilize instrument and reagent in the embedding reagent box to operate when preparing organoid frozen section, need not purchase the reagent alone again, makes things convenient for the experimenter to carry out the experiment operation.
The other technical scheme of the invention is as follows: an embedding method of a frozen section of organoid tissue comprises the following steps:
1) removing the culture medium in the cell culture hole on the culture hole plate, adding PBS buffer solution for three times to wash the residual culture medium, washing for 3min each time, adding methanol frozen at-20 deg.C for 2h into the cell culture hole, and fixing at room temperature for 20 min;
2) removing methanol in the cell culture hole, and then adding PBS buffer solution twice to wash residual methanol, wherein washing is carried out for 3min each time;
3) dyeing matrigel, adding methylene blue into the cell culture hole for dyeing, staying for 2min, removing the methylene blue, and washing residual washing by a PBS buffer solution;
4) inserting the gel taking device along the edge of the cell culture hole, taking out the organoids containing matrigel together, placing the organoids on the tinfoil paper of the bottom plate of the embedding box, and fixing the cylinder body of the embedding box on the bottom plate;
5) adding an OCT embedding medium from the opening of the cylinder body of the embedding box to ensure that the matrigel is completely covered by the OCT embedding medium, and then covering the cylinder cover;
6) placing the embedding box obtained in the step 5) into liquid nitrogen for quick freezing for 15min, and then transferring to a refrigerator with the temperature of 80 ℃ below zero for storage;
7) transferring the embedded sample obtained in the step 6) from a refrigerator at the temperature of-80 ℃ to an environment at the temperature of-20 ℃ for balancing for 3 hours, then slicing the embedded sample by adopting a freezing microtome according to the thickness of 10 mu L, and finally pasting the organoid slice on an anti-falling glass slide for preservation in the refrigerator at the temperature of-80 ℃.
Further, the volume of the methanol added in the step 1) is 3-5 times of the volume of the organoid.
Further, the volume of the OCT embedding medium in the step 5) is 2/3 of the embedding box.
Further, the PBS buffer was added 100. mu.L each time for washing.
Adopt above-mentioned technical scheme: the embedding method adopts methanol to fix a sample, can precipitate protein and saccharide in organoid cells, prevents the cells from autolysis, preserves the structural state of the cells before separation, prevents the organoid structure after separation from being damaged, adopts methylene blue to dye matrigel, is convenient for marking the position of the organoid sample, can slice the frozen sample according to the position with the dyeing when slicing the frozen sample, does not need to slice the position without the dyeing, thereby avoiding blind cutting, also avoiding searching the slice with the organoid from the slice with the blind cutting, greatly improving the success rate of preparing the organoid frozen slice, and improving the preparation efficiency.
The invention is further described with reference to the drawings and the specific embodiments in the following description.
Drawings
FIG. 1 is a schematic view of the structure of an embedding kit according to the present invention;
FIG. 2 is a schematic structural view of an embedding cassette according to the present invention;
FIG. 3 is a schematic view of the structure of the bottom plate of the cassette of the present invention;
FIG. 4 is a schematic structural view of the glue dispenser of the present invention;
FIG. 5 is a right side view of FIG. 3;
FIG. 6 is a schematic view showing the observation of HE staining of organoids in example 1 of the present invention;
FIG. 7 is a schematic view of multichannel fluorescence staining observation of an RNAscope of class 2 according to the embodiment of the present invention;
FIG. 8 is a schematic view of immunofluorescence staining of organoids according to example 3 of the present invention.
Detailed Description
Referring to fig. 1 to 8, an organoid tissue frozen section embedding kit, includes outer box 9, place an embedding box 11 in outer box 9, one get gluey ware 10 to and a set of embedding reagent 12, can set up a plurality of constant head tanks that are used for placing embedding box 11, getting gluey ware 9, embedding reagent 12 in outer box 9 respectively, the shape of each constant head tank can be according to embedding box 11, get gluey ware 10, embedding reagent 12 and design. Embedding box 11 includes barrel 1, bottom plate 2, the internal diameter of barrel 1 is 10 ~ 15mm, and barrel 1 is dark 15 ~ 20mm, 1 ~ 2mm of the wall thickness of barrel 1, and this barrel 1 can play moulding effect, moulds the shape that forms barrel 1 with sample and embedding medium jointly, and both freezing of being convenient for makes things convenient for the section again, makes final frozen section more regular. 2 upper ends of the bottom plate are provided with an annular positioning groove 3 for inserting the barrel body 1, the groove width of the annular positioning groove 3 is matched with the wall thickness of the barrel body 1, so that the barrel body 1 can be tightly clamped in the annular positioning groove 3 and fixed together with the bottom plate 2, a protection effect is achieved on a sample, the embedded sample is conveniently molded, the integral freezing treatment is achieved, and the purpose of matching the bottom support of the freezing slicer is achieved. The middle of the bottom plate 2 is provided with an axial through hole 4, and the inner diameter of the cylinder body 1 is the same as that of the axial through hole 4 of the bottom plate 2, so that a frozen sample in the embedding box can be taken out smoothly. The lower surface of bottom plate 2 bonds a fixed tin foil 5, the lower extreme through the sealed axial through hole 4 of tin foil 5, this tin foil 5 can prevent that the sample from freezing back and bottom plate 2 emergence adhesion, and the outward flange of 5 one sides of tin foil can extend bottom plate 2, and the part that extends does not take place to bond with bottom plate 2, tear tin foil 5 from this outward flange department when conveniently taking the frozen sample, when taking the frozen sample promptly, tear tin foil 5 gently, the last port from barrel 1 pushes away the sample lightly, can release the sample from the axial through hole 4 of bottom plate 2, thereby guarantee the integrality of organoid sample structure. The upper end of the barrel body 1 is provided with a barrel cover 6, the barrel cover 6 is connected with the barrel body 1 through a hinged portion, the barrel cover 6 can be prevented from being lost and is convenient to store, and the barrel cover 6 can be an outer cover or an inner plug. The upper surface of the cylinder cover 6 is frosted, so that the contents such as preparation date and the like can be written on the upper surface of the cylinder cover 6, and the required samples can be distinguished and identified conveniently in the later use.
Get gluey ware 10 and be equipped with handle 7, this handle 7 one end is cylindrical, and the other end is flat shape, handle 7 columniform one end can set up the annular knurl, plays anti-skidding effect, convenient operation. The flat-shaped end is vertically bent to form a glue taking part 8, the glue taking part 8 is used for taking a sample out of the culture pore plate and placing the sample on the tin foil 5 of the embedding box 11, the glue taking part 8 can be inserted along the edge of the cell culture pore of the culture pore plate, and as the culture environment of the organoid in the matrigel is a carbon dioxide incubator at 37 ℃, the matrigel in the cell culture pore is in a jelly shape, so that the glue taking part 8 can support the matrigel from the bottom of the cell culture pore by slightly rotating the handle 7, and the glue taking part 8 is attached to the bottom of the cell culture pore. Get the width of gluing portion 8 and be 3 ~ 3.5mm, highly be 3 ~ 4mm, can guarantee to get to glue portion 8 and have certain activity degree of freedom in the cell culture hole, after getting to glue portion 8 and hold matrigel, can hold handle 7 and make and get to glue portion 8 and control and rock to can the at utmost take out matrigel completely, avoid destroying the structure of organoid, guarantee the structural integrity of the organoid of taking out. Get extension end of gluing portion 8 and be circular-arc, the arc top height of circular arc is 1mm, makes to get gluey portion 8 and suits with the inner wall radian in cell culture hole, conveniently gets gluey portion 8 and removes in cell culture hole, further improves the integrality of taking out matrigel. The length of handle 7 is 150mm, and the diameter is the same with the width of getting gluey portion 8, and this length adapts to most experimenters' palm width, improves the comfort level of holding during the experiment.
The embedding reagent 12 includes methanol for immobilizing organoid proteins and antigens before embedding the mixture of matrigel and organoid, methylene blue for staining matrigel before embedding the mixture of matrigel and organoid, respectively, and an OCT embedding medium for embedding the mixture of matrigel and organoid,
taking a 96-hole culture well plate as an example, the width of the glue taking part 8 of the glue taking device is 3.5mm, the height is 4mm, the arc top height of the arc of the extending end of the glue taking part 8 is 1mm, the inner diameter of the cylinder 1 of the embedding box is 13.5mm, the depth of the cylinder 1 is 15mm, and the wall thickness of the cylinder 1 is 2 mm.
Embedding box 11, get gluey ware 10 and embedding reagent 12 and place in same embedding reagent box jointly, wherein methyl alcohol, methylene blue, the OCT embedding medium can be according to the quantity that the experiment needs, adorn respectively in different reagent bottles, for example according to the volume of the embedding box 11 of this embodiment, can be with 25ml methyl alcohol, 5ml methylene blue, 50ml OCT embedding medium and embedding box 11, it adorns in an embedding reagent box together to get gluey ware 10, then the experimenter is when preparing class organ frozen section, instrument and reagent in the embedding kit just can be directly utilized and operated, need not purchase reagent alone again, make things convenient for the experimenter to carry out experimental operation, and avoid experimental reagent's waste.
When the device is used, a sample 13 mixed with matrigel and organoid is taken out of a cell culture hole by a gel taking device, the sample is placed on the tin foil paper 5 of an embedding box, the barrel 1 of the embedding box is inserted into the annular positioning groove 3 of the bottom plate 2 to be clamped, the sample is embedded in the embedding box by the OCT embedding medium 14, the whole shaping is carried out by the shape of the barrel 1, the barrel cover 6 is covered, the whole embedding box embedded with the organoid is frozen to be stored, when the frozen sample needs to be taken, the barrel cover 6 is opened, the tin foil paper 5 on the lower end face of the bottom plate 2 is torn off, the frozen sample is slightly pushed from the upper end to be separated from the inner wall of the barrel 1, and then the frozen sample is pushed out from the lower end of the axial through hole 4 of the bottom plate 2.
To sum up, this embedding device simple structure, convenient to use, the completeness of assurance organoid structure that can the at utmost improves organoid frozen section's preparation success rate.
An embedding method of a frozen section of organoid tissue comprises the following steps:
1) removing the culture medium in the cell culture holes on the culture hole plate, sucking out the culture medium by using a pipette, adding PBS buffer solution for three times to wash the residual culture medium, adding 100 mu L of PBS buffer solution every time, wherein the washing time is 3min every time, then adding methanol frozen for 2h at the temperature of-20 ℃ into the cell culture holes, and fixing for 20min at room temperature to enable the organoid to enter a fixed state, wherein the adding volume of the methanol is 3-5 times of the volume of the organoid. After the organoid is separated from the body, the organoid is autolyzed due to the change of microenvironment, so that the structure of the organoid is destroyed, and the alcohol has the characteristic of strong penetrability and can well preserve the antigenicity of cells.
Thus, the addition of methanol can have the following effects: firstly, proteins and saccharides in organoid cells are precipitated, the action of a decomposition enzyme is stopped or reduced, autolysis is prevented, the structural state of the organoid before separation in vitro is preserved, the antigenicity of the organoid is preserved, and the antigen is not inactivated and dispersed; secondly, preserving protein, fat, glycogen, certain vitamins and pathological accumulation in the organoids, and maintaining the specificity characteristics of pathological changes; thirdly, the substances are converted into a state of being not easy to dissolve, and the dissolution and the loss caused by human factors in the process of preparing the frozen slices are prevented and reduced as much as possible; fourthly, the dyeing assisting function is achieved, and the subsequent dyeing is facilitated.
2) The methanol in the cell culture wells was removed, and then the residual methanol was washed by adding PBS buffer in two portions, each of which was 100. mu.L of PBS buffer, for 3 min.
3) The stroma glue is dyed, methylene blue is added into the cell culture holes for dyeing, the staying dyeing time is 2min, the methylene blue is removed, and the residual washing is washed by PBS buffer solution, because the stroma glue on the market is usually colorless or pale pink, and the culture time of the organoid in the stroma glue is more than 10 days, the mixture of the organoid and the stroma glue is transparent and colorless, the stroma glue is slightly dyed by the methylene blue, the position of the organoid sample is convenient to mark, the organoid sample can be sliced aiming at the colored position subsequently, and the preparation success rate of the frozen slice is ensured.
4) Inserting the gel taking device along the edge of the cell culture hole, taking out the matrigel and the organoid together, placing the matrigel and the organoid on the tin foil paper of the embedding box, and fixing the cylinder body of the embedding box on the bottom plate.
5) Adding an OCT embedding medium from the opening of the cylinder body of the embedding box to ensure that the matrigel is completely covered by the OCT embedding medium, then covering the cylinder cover, wherein the volume of the OCT embedding medium is 2/3 of the embedding box, so that the OCT embedding medium can completely cover the matrigel, and a certain vacancy is reserved in the cylinder body to ensure that a sample can be completely taken out after being frozen.
6) Placing the embedding box obtained in the step 5) into liquid nitrogen for quick freezing for 15min, and then transferring to a refrigerator with the temperature of 80 ℃ below zero for storage;
7) transferring the embedding box in the step 6) from a refrigerator at minus 80 ℃ to an environment at minus 20 ℃ for balancing for 3 hours to slightly dissolve the OCT embedding medium, smoothly taking out the frozen sample from the embedding box, slicing the sample according to the thickness of 10 mu m by using a freezing microtome, finally pasting the organoid slice on an anti-falling glass slide, storing the sliced organoid slice at minus 80 ℃, and carrying out protein detection within one year and RNA detection within 6 months.
Example 1, as shown in FIG. 6, the results of morphological staining (HE) using the organoid tissue frozen section embedding apparatus and embedding method of the present invention are as follows.
The lung organoids cultured for 15 days are removed from the culture medium, the organoids are embedded by the embedding method, the frozen sections are frozen according to the thickness of 10 mu m, the cut frozen sections are balanced for 20min at room temperature, washed three times by PBS (phosphate buffer solution) for 5min each time, stained by hematoxylin for 5min, washed by tap water, differentiated by hydrochloric acid and alcohol, washed by distilled water, stained by eosin for 5min, washed by distilled water, dried by blowing, transparent by xylene, sealed by neutral resin, and the pictures are collected by a stereomicroscope.
HE staining results: the spherical structure, solid sphere and hollow sphere of organoids can be observed, the structure is complete, the alveolar deconstruction is realized, and the envelope is complete.
Example 2 organoid tissue frozen section embedding apparatus and embedding method used to prepare organoid frozen sections as shown in fig. 7, RNAscope multichannel fluorescence assay was as follows.
The lung organoids cultured for 15 days are removed from the culture medium, organoid embedding is carried out by the embedding method, frozen sections are carried out according to the thickness of 10 mu m, the cut organoid sections are baked for 30min at 60 ℃, fixed again for 15min by 4 percent paraformaldehyde, treated for 5min by 50 percent, 75 percent and 100 percent of ethanol in turn, and sample repair, probe hybridization, fluorescent dye combination, DAPI nuclear staining, anti-fluorescence quenching machine sealing and laser confocal image acquisition are carried out in turn according to the ACD brand RNAscope multichannel fluorescence detection kit steps. The RNAscope multichannel fluorescence detection result shows that: the expression of in-situ RNA genes on single cells can be observed, the staining results are scattered or clustered fluorescent spots which are typical in-situ RNA hybridization staining results, and the organoid frozen section prepared by the kit can prevent the RNA degradation of a sample and ensure the integrity of RNA fragments.
Example 3, as shown in FIG. 8, the results of immunofluorescent staining experiments using the organoid tissue frozen section embedding apparatus and method are as follows.
Removing a culture medium from a lung organoid cultured for 15 days, carrying out organoid embedding by using the embedding method of the invention, carrying out frozen section according to the thickness of 10 mu m, washing 3 times by PBS, perforating for 10min, sealing donkey serum for 1h, adding primary antibody (SPC, T1 alpha) for overnight incubation at 4 ℃, balancing for 30min on the next day and room temperature, washing 3 times by PBS for 5min each time, adding secondary antibodies (donkey anti-mouse 488 and donkey anti-rabbit 594) for incubation for 1h at 37 ℃, washing by PBS, staining nuclei by DAPI, sealing an anti-fluorescence quencher, and collecting pictures by laser confocal focusing. Immunofluorescence results: the specific expression of SPC and T1 alpha can be observed, the organoid structural morphology is complete, and the organoid frozen section prepared by the embedding device and the embedding method can well keep the protein of a sample and prevent the antigen from losing.
The embedding method adopts methanol to fix a sample, can precipitate protein and saccharide in organoid cells, prevents the cells from autolysis, preserves the structural state of the organoid before separation, prevents the organoid structure after separation from being damaged, adopts methylene blue to dye matrigel, is convenient for marking the position of the organoid sample, can slice the frozen sample according to the position with the dyeing when slicing the frozen sample, does not need to slice the position without the dyeing, thereby avoiding blind cutting, also avoiding searching the slice with the organoid from the slice with the blind cutting, greatly improving the success rate of preparing the organoid frozen slice, and improving the preparation efficiency.
Claims (10)
1. The utility model provides an organoid tissue frozen section embedding kit, outer box (9), its characterized in that: an embedding box (11), a glue taking device (10) and a group of embedding reagents (12) are placed in the outer box (9), the embedding box comprises a barrel body (1) and a bottom plate (2), the inner diameter of the barrel body (1) is 10-15 mm, the depth of the barrel body (1) is 15-20 mm, the wall thickness of the barrel body (1) is 1-2 mm, an annular positioning groove (3) for inserting the barrel body (1) is formed in the upper end of the bottom plate (2), the groove width of the annular positioning groove (3) is matched with the wall thickness of the barrel body (1), an axial through hole (4) is formed in the middle of the bottom plate (2), a tin foil paper (5) is fixedly bonded to the lower surface of the bottom plate (2), the lower end of the axial through hole (4) is sealed through the tin foil paper (5), and a barrel cover (6) is arranged at the upper end of the barrel body (1);
the glue taking device is provided with a handle (7), one end of the handle (7) is cylindrical, the other end of the handle (7) is flat, the flat end is vertically bent to form a glue taking part (8), the width of the glue taking part (8) is 3-3.5 mm, the height of the glue taking part (8) is 3-4 mm, and the glue taking part (8) is used for taking out a sample from a culture pore plate and placing the sample on the tin foil paper (5); the embedding reagent (12) comprises methanol, methylene blue and an OCT embedding agent.
2. The organoid tissue frozen section embedding kit according to claim 1, wherein: the extension end of the glue taking part (8) is arc-shaped, and the arc top height of the arc is 1 mm.
3. The organoid tissue frozen section embedding kit according to claim 1, wherein: the length of the handle (7) is 150mm, and the diameter of the handle is the same as the width of the glue taking part (8).
4. The organoid tissue frozen section embedding kit according to claim 1, wherein: the cylinder cover (6) is connected with the cylinder body (1) through a hinged part.
5. The organoid tissue frozen section embedding kit according to claim 1, wherein: the upper surface of the cylinder cover (6) is frosted.
6. The organoid tissue frozen section embedding kit according to claim 1, wherein: the inner diameter of the cylinder body (1) is the same as that of the axial through hole (4) of the bottom plate (2).
7. An embedding method of a frozen section of a organoid tissue is characterized by comprising the following steps:
1) removing the culture medium in the cell culture hole on the culture hole plate, adding PBS buffer solution for three times to wash the residual culture medium, washing for 3min each time, adding methanol frozen at-20 deg.C for 2h into the cell culture hole, and fixing at room temperature for 20 min;
2) removing methanol in the cell culture hole, and then adding PBS buffer solution twice to wash residual methanol, wherein washing is carried out for 3min each time;
3) dyeing matrigel, adding methylene blue into the cell culture hole for dyeing, staying for 2min, removing the methylene blue, and washing residual washing by a PBS buffer solution;
4) inserting the gel taking device along the edge of the cell culture hole, taking out the organoids containing matrigel together, placing the organoids on the tinfoil paper of the bottom plate of the embedding box, and fixing the cylinder body of the embedding box on the bottom plate;
5) adding an OCT embedding medium from the opening of the cylinder body of the embedding box to ensure that the matrigel is completely covered by the OCT embedding medium, and then covering the cylinder cover;
6) placing the embedding box obtained in the step 5) into liquid nitrogen for quick freezing for 15min, and then transferring to a refrigerator with the temperature of 80 ℃ below zero for storage;
7) transferring the embedded sample obtained in the step 6) from a refrigerator at the temperature of-80 ℃ to an environment at the temperature of-20 ℃ for balancing for 3 hours, then slicing the embedded sample by adopting a freezing microtome according to the thickness of 10 mu L, and finally pasting the organoid slice on an anti-falling glass slide for preservation in the refrigerator at the temperature of-80 ℃.
8. The method for embedding organoid tissue frozen sections according to claim 6, wherein: the volume of the methanol added in the step 1) is 3-5 times of the volume of the organoid.
9. The method for embedding organoid tissue frozen sections according to claim 6, wherein: the volume of the OCT embedding medium in the step 5) is 2/3 of the embedding box.
10. The method for embedding organoid tissue frozen sections according to claim 6, wherein: the PBS buffer was added 100. mu.L each time for washing.
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