CN113340699B - 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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- CN113340699B CN113340699B CN202110611852.4A CN202110611852A CN113340699B CN 113340699 B CN113340699 B CN 113340699B CN 202110611852 A CN202110611852 A CN 202110611852A CN 113340699 B CN113340699 B CN 113340699B
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/36—Embedding or analogous mounting of samples
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
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- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
- G01N2001/2873—Cutting or cleaving
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Abstract
The invention relates to a frozen section embedding kit and an embedding method for a similar organ tissue, which can improve the structural integrity of the similar organ, reduce the loss of protein antigens, prevent the degradation of genes (RNA and DNA) and greatly improve the preparation success rate of the frozen section of the similar organ tissue.
Description
Technical Field
The invention relates to the technical field of organoids, in particular to a frozen embedding kit for organoids and an embedding method.
Background
Organoids belong to three-dimensional (3D) cell cultures and contain some of the key properties that represent organs, as proposed by Clever et al in "Sato T,Vries RG,Snippert HJ,et al.Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche"Nature.2009May14;459(7244):262-5, for isolated culture of small intestine stem cells, where it was found that a single cell could form the crypt villus structure of the intestine. In recent years, the organoid technology has been developed rapidly, and various organoids such as brain, lung, liver, kidney, intestinal tract, stomach and the like are established successively, so that the organoid is applied to a plurality of fields such as accurate medical treatment, organ transplantation, drug screening, drug action mechanism research and the like. The organoids have the advantages of simulating signal communication among various cells and movement of cells in a body, maintaining gene stability in the culture process, simulating development of human body and disease, making up the defects of animal experiments, and realizing real-time imaging.
Cell growth in vitro relies on cell adhesion and cytoskeleton, and since organoids are three-dimensional structures, support from the corresponding environment is required, and the most common approach to promote organoid formation into three-dimensional properties is to use a solid extracellular matrix that supports cell growth and cell adhesion. Currently, the commonly used extracellular matrices are mainly three types of natural extracellular matrix, matrigel (also called organism-derived matrix), and extracellular matrix-chemical hydrogel, wherein Matrigel is one of the most widely used types.
Matrigel has the characteristics of presenting different phases at different temperatures, is solid when being below-20 ℃, is liquid when being between 4 and 8 ℃, is solidified into jelly shape at more than 37 ℃, and the culture environment of organoids in matrigel is a carbon dioxide incubator at 37 ℃, so that organoids fixed in matrigel are jelly shape, the special phases cause very difficult preparation of frozen slices directly in organoid matrigel, and the main problems are as follows:
first, organoid has many kinds of embedding and fixing agents, and various embedding and fixing agents have different use conditions, so that the antigen cannot be lost and the gene cannot be degraded.
Secondly, the matrigel color problem, because matrigel itself does not have the colour, consequently can't confirm the position of organoid after fixed with embedding fixed reagent, can only blindly cut, hardly obtain fine sample position, extravagant experiment consumptive material even can lead to the experiment failure.
Thirdly, the types of the organoid culture pore plates are numerous, the organoids can be cultured from 6 pore plates to 96 pore plates, the sizes of the culture pores of different pore plates are different, and the matrigel is jelly-shaped, so that the matrigel for culturing the organoids is difficult to completely take out, and the structural damage of the organoids is easy to cause.
Fourth, since organoids are basically cultured in cell culture plates, the cell culture plates cannot be directly embedded, and the number of cultured organoids varies from plate to plate, and there is no good embedding mold.
Therefore, in the preparation of frozen section samples of organoids, the fixation of organoid tissues, the sampling of organoid tissues and the embedding of organoid tissues into a mold are all key factors which lead to the success of the preparation of frozen sections of organoids, and are also urgent problems to be solved.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides an organ-like tissue frozen section embedding kit and an embedding method, which can improve the structural integrity of organs, reduce the loss of protein antigens, prevent the degradation of genes (RNA and DNA) and greatly improve the preparation success rate of organ-like tissue frozen sections.
The technical scheme of the invention is as follows: the utility model provides an organ tissue frozen section embedding kit, includes the outer box, be equipped with an embedding box, a glue taker in the outer box to and a set of embedding reagent, the embedding box includes barrel, bottom plate, the internal diameter of barrel is 10 ~ 15mm, and barrel depth 15 ~ 20mm, the wall thickness of barrel is 1 ~ 2mm, the bottom plate upper end sets up one and supplies barrel male annular positioning groove, annular positioning groove's slot width coincides with the wall thickness of barrel, be equipped with axial through-hole in the middle of the bottom plate, the lower surface bonding of bottom plate is fixed a tinfoil paper, the lower extreme of axial through-hole is sealed through the tinfoil paper, the barrel upper end is equipped with the cover;
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 a culture pore plate and placing the sample on tin foil paper; the embedding reagent comprises methanol, methylene blue and an OCT embedding agent.
Furthermore, the extending end of the glue taking part is arc-shaped, and the arc top height of the arc is 1mm.
Further, the length of the handle is 150mm, and the diameter is the same as the width of the glue taking part.
Further, the cylinder cover and the cylinder body are connected together through a hinge part.
Further, the upper surface of the cylinder cover is frosted.
Further, the inner diameter of the cylinder body is the same as the inner diameter of the axial through hole of the bottom plate.
The technical scheme is adopted: the flat end of the glue taking device handle is vertically bent to form a glue taking part, so that the glue taking part can be inserted along the edge of a cell culture hole of a culture hole plate, and as matrigel in the cell culture hole is jelly-shaped, matrigel can be supported at the bottom of the cell culture hole through the glue taking part, the width of the glue taking part is 3-3.5 mm, the height is 3-4 mm, a certain degree of freedom of movement of the glue taking part in the cell culture hole can be ensured, and after the matrigel is supported by the glue taking part, the handle can be held to enable the glue taking part to shake left and right, so that matrigel can be completely taken out to the greatest extent, the damage to the structure of organoids can be prevented, and the structural integrity of the organoids taken out can be ensured. The matrigel taken out by the gel taking device is placed on the tinfoil paper of the embedding box, the barrel of the embedding box is inserted into the annular positioning groove of the bottom plate to be fixed, so that the organoid is embedded in the barrel, the organoid sample after OCT embedding is conveniently frozen and matched with the collet of the frozen microtome in whole by shaping the shape of the barrel, and the embedded sample can be integrally taken out due to the fact that the tinfoil paper is fixedly adhered to the lower surface of the bottom plate, when the frozen sample is sliced, the tinfoil paper is torn off, the sample is gently pushed from the upper port of the barrel, and the sample can be pushed out from the axial through hole of the bottom plate, so that the structural integrity of the organoid is ensured. The embedding kit has the advantages of simple structure and convenient use, can ensure the integrity of the organoid structure to the greatest extent, and improves the preparation success rate of the frozen organoid slice. And, because embedding reagent and embedding box, get gluey ware and adorn in an embedding kit together, experimenter just can directly utilize instrument and reagent in the embedding kit to operate when preparing organoid section frozen, need not purchase reagent alone again, make things convenient for experimenter to carry out experimental operation.
The other technical scheme of the invention is as follows: an embedding method of frozen sections of the organoid tissues comprises the following steps:
1) Removing the culture medium in the cell culture holes on the culture hole plate, then adding PBS buffer solution for cleaning the residual culture medium for three times, cleaning for 3min each time, adding methanol frozen for 2h at-20 ℃ into the cell culture holes, and fixing for 20min at room temperature;
2) Removing methanol in the cell culture wells, and then adding PBS buffer solution twice to clean residual methanol for 3min each time;
3) Dyeing matrigel, adding methylene blue into a cell culture hole for dyeing, keeping the dyeing time for 2min, removing the methylene blue, and cleaning the residual by using PBS buffer solution;
4) Inserting the gel taker along the edge of the cell culture hole, taking out the organoids containing matrigel, placing the organoids on the tinfoil paper of the bottom plate of the embedding box, and fixing the barrel of the embedding box on the bottom plate;
5) Adding an OCT embedding agent from a cylinder opening of the embedding box, so that the matrigel is completely covered by the OCT embedding agent, and then covering a cylinder cover;
6) Placing the embedding box in the step 5) into liquid nitrogen, freezing for 15min, and then transferring to a refrigerator at the temperature of minus 80 ℃ for preservation;
7) Transferring the sample embedded in the step 6) from a refrigerator at the temperature of minus 80 ℃ to an environment at the temperature of minus 20 ℃ for balancing for 3 hours, slicing the sample by adopting a frozen microtome according to the thickness of 10 mu L, and finally pasting the organoid slice on an anti-drop glass slide and preserving the organoid slice in the refrigerator at the temperature of minus 80 ℃.
Further, the methanol is added in the step 1) in a volume 3 to 5 times the volume of the organoid.
Further, the volume of OCT embedding agent in step 5) is 2/3 of the volume of the embedding cassette.
Further, the PBS buffer was washed with 100. Mu.L of each buffer.
The technical scheme is adopted: the embedding method adopts methanol to fix the sample, can lead proteins and saccharides in organoid cells to be precipitated, prevent the cells from autolyzing, preserve the structural state before the cells are isolated, prevent the organoid structure after the cells are isolated from being damaged, and the embedding method also adopts methylene blue to dye matrigel, thereby being convenient for marking the position of the organoid sample, and when the frozen sample is sliced, the slice can be carried out aiming at the position with the dye, and the slice is not needed at the position without the dye, thereby avoiding blind cutting, and also avoiding the need of searching the slice with organoid from the slice with the blind cutting, greatly improving the preparation success rate of the frozen organoid slice and improving the preparation efficiency.
The invention is further described below with reference to the drawings and specific examples.
Drawings
FIG. 1 is a schematic diagram of the structure of an embedding kit of the present invention;
FIG. 2 is a schematic view of the structure of the embedding cassette of 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 view of the structure of the adhesive dispenser of the present invention;
FIG. 5 is a right side view of FIG. 3;
FIG. 6 is a schematic representation of HE staining observations of organoids according to example 1 of the present invention;
FIG. 7 is a schematic representation of the multi-channel fluorescent staining of organoid RNAscope of example 2 of the present invention;
FIG. 8 is a schematic diagram showing immunofluorescent staining of organoids according to example 3 of the present invention.
Detailed Description
Referring to fig. 1 to 8, an embedding kit for frozen sections of organoid tissues comprises an outer box 9, wherein an embedding box 11, a glue taking device 10 and a group of embedding reagents 12 are placed in the outer box 9, a plurality of positioning grooves for placing the embedding box 11, the glue taking device 10 and the embedding reagents 12 can be respectively arranged in the outer box 9, and the shape of each positioning groove can be designed according to the embedding box 11, the glue taking device 10 and the embedding reagents 12. The embedding box 11 comprises a barrel 1 and a bottom plate 2, wherein the inner diameter of the barrel 1 is 10-15 mm, the depth of the barrel 1 is 15-20 mm, the wall thickness of the barrel 1 is 1-2 mm, the barrel 1 can play a role in shaping, and the sample and the embedding agent are jointly shaped into the barrel 1, so that the freezing is convenient, the slicing is convenient, and the final frozen slicing is more regular. The upper end of the bottom plate 2 is provided with an annular positioning groove 3 for the insertion of the cylinder body 1, the groove width of the annular positioning groove 3 is matched with the wall thickness of the cylinder body 1, so that the cylinder body 1 can be tightly clamped in the annular positioning groove 3 and fixed with the bottom plate 2, the sample is protected, the embedded sample is conveniently shaped, the whole freezing treatment is realized, and the purpose of matching the bottom support of the frozen slicer is achieved. An axial through hole 4 is arranged in the middle of the bottom plate 2, and the inner diameter of the cylinder body 1 is the same as the inner diameter of the axial through hole 4 of the bottom plate 2, so that frozen samples in the embedding box can be taken out smoothly. The lower surface bonding of bottom plate 2 is fixed a tinfoil 5, through the lower extreme of the sealed axial through-hole 4 of tinfoil 5, this tinfoil 5 can prevent that the sample from freezing the back and take place the adhesion with bottom plate 2, and the outward flange of tinfoil 5 one side can extend bottom plate 2, and the part that extends does not take place the bonding with bottom plate 2, tear tinfoil 5 from this outward flange department when conveniently taking frozen sample, when taking frozen sample promptly, tear tinfoil 5 gently, the upper port from barrel 1 gently pushes the sample, can follow the sample and release in the axial through-hole 4 of bottom plate 2 to guarantee organoid sample structure's integrality. The barrel 1 upper end is equipped with barrel cover 6, barrel cover 6 is in the same place through articulated portion connection with barrel 1, can prevent that barrel cover 6 from losing, conveniently accomodates, and this barrel cover 6 can be the enclosing cover, also can set up the interior stopper. The upper surface of the cylinder cover 6 is frosted, so that contents such as preparation date and the like can be written on the upper surface of the cylinder cover 6, and the sample required by identification can be distinguished quickly during later use.
The glue taking device 10 is provided with a handle 7, one end of the handle 7 is cylindrical, the other end of the handle 7 is flat, and knurling can be arranged on one cylindrical end of the handle 7, so that the glue taking device has an anti-skid function and is convenient to operate. The flat end is vertically bent to form a glue taking part 8, the glue taking part 8 is used for taking out a sample from a culture pore plate and placing the sample on the tinfoil paper 5 of the embedding box 11, the glue taking part 8 can be inserted along the edge of a cell culture pore of the culture pore plate, and as the culture environment of organoids in matrigel is a carbon dioxide incubator at 37 ℃, matrigel in the cell culture pore is in a jelly shape, 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. The width of the glue taking part 8 is 3-3.5 mm, the height is 3-4 mm, a certain degree of freedom of movement of the glue taking part 8 in the cell culture hole can be ensured, after the glue taking part 8 holds the matrigel, the handle 7 can be held to enable the glue taking part 8 to shake left and right, so that the matrigel can be completely taken out to the greatest extent, the damage to the structure of the organoid is avoided, and the structural integrity of the taken organoid is ensured. The extension end of the glue taking part 8 is arc-shaped, the arc top of the arc is 1mm, so that the glue taking part 8 is adapted to the radian of the inner wall of the cell culture hole, the glue taking part 8 can conveniently move in the cell culture hole, and the integrality of the matrix glue taken out is further improved. The length of the handle 7 is 150mm, the diameter is the same as the width of the glue taking part 8, the length is suitable for the palm width of most experimenters, and the holding comfort level in the experiment is improved.
The embedding reagent 12 includes methanol for immobilizing the organoid protein and antigen before embedding the mixture of matrigel and organoid, methylene blue for staining matrigel before embedding the mixture of matrigel and organoid, and OCT embedding agent for embedding the mixture of matrigel and organoid, respectively.
Taking a 96-hole culture pore plate as an example, the width of a 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 a barrel 1 of the embedding box is 13.5mm, the depth of the barrel 1 is 15mm, and the wall thickness of the barrel 1 is 2mm.
The embedding box 11, the glue taking device 10 and the embedding reagent 12 are placed in the same embedding reagent box together, wherein the methanol, the methylene blue and the OCT embedding reagent can be respectively arranged in different reagent bottles according to the quantity required by experiments, for example, according to the volume of the embedding box 11 of the embodiment, 25ml of methanol, 5ml of methylene blue and 50ml of OCT embedding reagent can be arranged in one embedding reagent box together with the embedding box 11 and the glue taking device 10, so that an experimenter can directly use tools and reagents in the embedding reagent box to operate when preparing the frozen slices of the organoids, the experimenter does not need to purchase the reagents independently, the experimenter can conveniently perform experimental operation, and the waste of the experimental reagent is avoided.
When the device is used, firstly, a glue taking device is used for taking out a sample 13 mixed by matrigel and organoid from a cell culture hole, the sample is placed on a tinfoil paper 5 of an embedding box, then a barrel 1 of the embedding box is inserted into an annular positioning groove 3 of a bottom plate 2 to be clamped, the sample is embedded in the embedding box by using an OCT embedding agent 14, the whole shaping is carried out through the shape of the barrel 1, a barrel cover 6 is covered, then the whole embedding box embedded with organoid is frozen and preserved, when the frozen sample is needed to be taken, the barrel cover 6 is opened, the tinfoil paper 5 on the lower end surface of the bottom plate 2 is torn off, the frozen sample is slightly pushed from an upper port to be separated from the inner wall of the barrel 1, and then the frozen sample is pushed out from the lower end of an axial through hole 4 of the bottom plate 2.
In conclusion, the embedding device has simple structure and convenient use, can ensure the integrity of the organoid structure to the greatest extent, and improves the preparation success rate of the frozen organoid slice.
An embedding method of frozen sections of the organoid tissues comprises the following steps:
1) Removing the culture medium in the cell culture holes on the culture hole plate, sucking the culture medium out by a liquid transfer device, then adding PBS buffer solution three times to clean the residual culture medium, adding 100 mu L of PBS buffer solution each time, wherein the cleaning time is 3min, adding methanol frozen for 2h at the temperature of minus 20 ℃ into the cell culture holes, and fixing for 20min at room temperature to enable the organoids to enter a fixed state, wherein the adding volume of the methanol is 3-5 times of the organoids. After the organoids are isolated, the change of microenvironment can cause the organoids to autolyze, so that the structure of the organoids is destroyed, and the alcohols have the characteristic of strong penetrability, so that the antigenicity of cells can be well preserved.
Thus, the addition of methanol may have the following effects: firstly, proteins and saccharides in organoid cells are precipitated, the action of a degrading enzyme is stopped or reduced, autolysis is prevented, the structural state of the organoid before in vitro is preserved, the antigenicity of the organoid is preserved, and the antigen is not inactivated and is not dispersed; second, preserving proteins, fat, glycogen, certain vitamins and pathological accumulations within organoids, maintaining the specific characteristics of lesions; thirdly, the substances are converted into an insoluble state, so that dissolution and loss caused by human factors in the process of preparing frozen slices are prevented and reduced as much as possible; fourth, the dyeing auxiliary function is achieved, and the subsequent dyeing is facilitated.
2) The cell culture wells were cleared of methanol, and then the remaining methanol was washed by adding PBS buffer in two portions, 100. Mu.L of PBS buffer each time, and 3min each time.
3) And (3) dyeing the matrigel, adding methylene blue into a cell culture hole for dyeing, keeping the dyeing time for 2min, removing the methylene blue, and cleaning the residual cleaning by using a PBS buffer solution, wherein the matrigel on the market is usually colorless or light pink, and the culture time of the organoids in the matrigel is more than 10 days, so that the mixture of the organoids and the matrigel is transparent and colorless, the matrigel is slightly dyed by adopting the methylene blue, the positions of organoid samples are conveniently marked, and then, slicing can be performed on the colored positions, thereby ensuring the preparation success rate of frozen slices.
4) The matrigel and organoid are taken out together by inserting the gel taking device along the edge of the cell culture hole, and are placed on the tinfoil paper of the embedding box, and then the barrel of the embedding box is fixed on the bottom plate.
5) The OCT embedding agent is added from the cylinder mouth of the embedding box, so that the matrigel is completely covered by the OCT embedding agent, then the cylinder cover is covered, the volume of the OCT embedding agent is 2/3 of that of the embedding box, so that the OCT embedding agent can completely cover the matrigel, and a certain vacancy is reserved in the cylinder, and the sample can be completely taken out after the sample is frozen.
6) Placing the embedding box in the step 5) into liquid nitrogen, freezing for 15min, and then transferring to a refrigerator at the temperature of minus 80 ℃ for preservation;
7) Transferring the embedding box in the step 6) from a refrigerator at the temperature of minus 80 ℃ to an environment at the temperature of minus 20 ℃ for balancing for 3 hours, slightly dissolving the OCT embedding agent, smoothly taking out the frozen sample from the embedding box, slicing the frozen sample according to the thickness of 10 mu m by adopting a frozen microtome, finally attaching the organoid slice to an anti-drop glass slide, and storing the cut organoid slice at the temperature of minus 80 ℃ for protein detection within one year and RNA detection within 6 months.
Example 1 as shown in fig. 6, morphological staining (HE) results were performed using the organoid tissue frozen section embedding apparatus and embedding method of the present invention as follows.
The lung organoids cultured for 15 days are removed the culture medium, the organoids are embedded by the embedding method of the invention, frozen sections are carried out according to the thickness of 10 mu m, the cut frozen sections are balanced for 20min at room temperature, washed three times by PBS, each time for 5min, stained with hematoxylin for 5min, then rinsed with tap water, differentiated by hydrochloric acid and alcohol, washed by distilled water, stained with eosin for 5min, washed by distilled water, dried by blow, transparent by xylene, sealed by neutral resin, and pictures are collected by a stereoscopic microscope.
HE staining results: the sphere structure of the organoids, solid spheres and hollow spheres, are observed, the structure is complete, alveolar-like deconfiguration is achieved, and the envelope is complete.
Example 2, as shown in fig. 7, organoid frozen sections were prepared using the organoid tissue frozen section embedding apparatus and embedding method, and RNAscope multichannel fluorescence detection was as follows.
The lung organoids cultured for 15 days are removed the culture medium, organoid embedding is carried out by the embedding method of the invention, frozen sections are carried out according to the thickness of 10 mu m, the cut organoids sections are baked for 30min at 60 ℃, are fixed for 15min again by 4% paraformaldehyde, are treated for 5min by 50%,75% and 100% ethanol in sequence, and are subjected to sample restoration, probe hybridization, fluorescent dye combination, DAPI nuclear dyeing, anti-fluorescent quenching machine sealing and laser confocal acquisition according to the steps of ACD brand RNAscope multichannel fluorescence detection kit. The RNAscope multichannel fluorescence detection result shows that: the expression of the in-situ RNA genes on single cells can be observed, the dyeing results are scattered or clustered fluorescent spots, and are typical in-situ RNA hybridization dyeing results, and the organoid frozen section prepared by the kit can prevent the degradation of sample RNA and ensure the integrity of RNA fragments.
Example 3, as shown in fig. 8, the following immunofluorescence staining test results were obtained using the organoid tissue frozen section embedding apparatus and embedding method.
The lung organoids cultured for 15 days are removed the culture medium, organoid embedding is carried out by the embedding method of the invention, frozen sections are carried out according to the thickness of 10 mu m, PBS is washed 3 times, cells are perforated for 10min, donkey serum is blocked for 1h, primary antibody (SPC, T1 alpha) is added for overnight incubation at 4 ℃, the next day, room temperature is balanced for 30min, PBS is washed 3 times, each time is 5min, secondary antibody (donkey anti-mouse 488, donkey anti-rabbit 594) is added for incubation at 37 ℃ for 1h, PBS is washed, DAPI is used for nuclear staining, anti-fluorescence quenching machine sealing sheet, and laser confocal is carried out to collect pictures. Immunofluorescence results: the specific expression of SPC and T1 alpha can be observed, the structure and shape of the organoid are complete, and the organoid frozen section prepared by the embedding device and the embedding method can well keep sample proteins and prevent antigen loss.
The embedding method adopts methanol to fix the sample, can lead proteins and saccharides in organoid cells to be precipitated, prevent cells from autolyzing, preserve the structural state before the organoid is isolated, prevent the organoid structure after the organoid is isolated from being damaged, and the embedding method also adopts methylene blue to dye matrigel, thereby being convenient for marking the position of the organoid sample, and when the frozen sample is sliced, the slice can be carried out aiming at the position with the dye, and the position without the dye does not need to be sliced, thereby avoiding blind cutting, and also avoiding the need to search the slice with organoid from the slice with the blind cutting, greatly improving the preparation success rate of the frozen organoid slice and improving the preparation efficiency.
Claims (9)
1. An organ-like tissue frozen section embedding kit, outer box (9), 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 cylinder body (1) and a bottom plate (2), the inner diameter of the cylinder body (1) is 10-15 mm, the depth of the cylinder body (1) is 15-20 mm, the wall thickness of the cylinder body (1) is 1-2 mm, an annular positioning groove (3) for the insertion of the cylinder body (1) is arranged at 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 cylinder body (1), an axial through hole (4) is arranged in the middle of the bottom plate (2), a tinfoil 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 by the tinfoil paper (5), and a cylinder cover (6) is arranged at the upper end of the cylinder 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 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 is 3-4 mm, the extending end of the glue taking part (8) is arc-shaped, the arc top of the arc is 1mm, and the glue taking part (8) is used for taking out a sample from a culture pore plate and placing the sample on tinfoil paper (5); the embedding reagent (12) comprises methanol, methylene blue and an OCT embedding agent.
2. The organoid tissue frozen section embedding kit of claim 1, wherein: the length of the handle (7) is 150mm, and the diameter is the same as the width of the glue taking part (8).
3. The organoid tissue frozen section embedding kit of claim 1, wherein: the cylinder cover (6) is connected with the cylinder body (1) through a hinge part.
4. The organoid tissue frozen section embedding kit of claim 1, wherein: the upper surface of the cylinder cover (6) is frosted.
5. The organoid tissue frozen section embedding kit of claim 1, wherein: the inner diameter of the cylinder body (1) is the same as the inner diameter of the axial through hole (4) of the bottom plate (2).
6. A method of embedding frozen organoid tissue sections using the kit of claim 1, characterized by the steps of:
1) Removing the culture medium in the cell culture holes on the culture hole plate, then adding PBS buffer solution for cleaning the residual culture medium for three times, cleaning for 3min each time, adding methanol frozen for 2h at-20 ℃ into the cell culture holes, and fixing for 20min at room temperature;
2) Removing methanol in the cell culture wells, and then adding PBS buffer solution twice to clean residual methanol for 3min each time;
3) Dyeing matrigel, adding methylene blue into a cell culture hole for dyeing, keeping the dyeing time for 2min, removing the methylene blue, and cleaning the residual by using PBS buffer solution;
4) Inserting the gel taker along the edge of the cell culture hole, taking out the organoids containing matrigel, placing the organoids on the tinfoil paper of the bottom plate of the embedding box, and fixing the barrel of the embedding box on the bottom plate;
5) Adding an OCT embedding agent from a cylinder opening of the embedding box, so that the matrigel is completely covered by the OCT embedding agent, and then covering a cylinder cover;
6) Placing the embedding box in the step 5) into liquid nitrogen, freezing for 15min, and then transferring to a refrigerator at the temperature of minus 80 ℃ for preservation;
7) Transferring the sample embedded in the step 6) from a refrigerator at the temperature of minus 80 ℃ to an environment at the temperature of minus 20 ℃ for balancing for 3 hours, slicing the sample by adopting a frozen microtome according to the thickness of 10 mu L, and finally pasting the organoid slice on an anti-drop glass slide and preserving the organoid slice in the refrigerator at the temperature of minus 80 ℃.
7. The method of embedding frozen organoid tissue sections in accordance with claim 6, wherein: the volume of methanol added in the step 1) is 3-5 times of the volume of the organoid.
8. The method of embedding frozen organoid tissue sections in accordance with claim 6, wherein: the volume of OCT embedding agent in step 5) is 2/3 of the volume of the embedding cassette.
9. The method of embedding frozen organoid tissue sections in accordance with claim 6, wherein: the PBS buffer was washed with 100. Mu.L of each buffer.
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