A method for immunofluorescence staining of organoids and a method for three-dimensional reconstruction

Abstract

This invention relates to a method for immunofluorescence staining and three-dimensional reconstruction of organoids, belonging to the field of cell biology technology. The method includes the following steps: collecting cultured organoids; taking the organoids at a magnification of 40-60x; sequentially fixing, permeabilizing, and blocking them; then incubating them sequentially with primary and secondary antibody solutions; finally, staining the nuclei; and mounting the slides after staining. This invention avoids the tissue detachment problem that may occur when using climbing slides and paraffin sections for immunofluorescence staining of three-dimensional structures such as organoids; the entire process maintains the structural integrity of the three-dimensional structures of organoids, facilitating three-dimensional reconstruction; different numbers of organoids can be stained according to their size and requirements, ensuring thorough staining; staining is performed directly under a stereomicroscope, requiring only one drop of antibody solution each time, saving reagents and antibodies; multiple combinations of staining can be performed on multiple organoids at once, or multiple staining methods can be applied to a single organoid.

Description

Technical Field

[0001] This invention relates to the field of cell biology technology, specifically to a method for immunofluorescence staining and three-dimensional reconstruction of organoids. Background Technology

[0002] Immunofluorescence technique is one of the earliest developed labeling immunoassay techniques. It is a technique built upon immunology, biochemistry, and microscopy. This method binds antibody molecules to tracer substances and utilizes antigen-antibody reactions to locate antigens within tissues or cells.

[0003] Immunofluorescence staining methods mainly include: (1) centrifugation; the drawback is that centrifugation will cause some cell clusters to be lost, cells to be damaged, scattered and deformed, and the cells inside the cell clusters will not be stained sufficiently. (2) staining with a staining device; the drawback is that the residual liquid is not completely removed or the cell clusters are easy to dry out, and one device can only process one set of samples, wasting reagents and antibodies. If the classic paraffin embedding technique is used directly for immunofluorescence staining of organoids and other three-dimensional structures after embedding, problems such as poor staining effect, dark background or no staining, and cell cluster sections are easy to fall off the slide will occur; in addition, organoids and other three-dimensional structures are small, only 100-200um, which requires high embedding and sectioning techniques.

[0004] There have been some new research advances in immunofluorescence staining methods. For example, Chinese patent CN101329230B discloses an improved immunofluorescence cell staining method, including five steps: fixation, permeabilization, surface staining and nuclear staining, washing, and resuspending. This is an improvement on the Foxp3 immunofluorescence cell staining method from eBioscience, changing the two-step multiplex staining method to a one-step multiplex staining method, simultaneously performing cell surface and nuclear staining. Chinese patent CN101226118B discloses a cytochemical staining method compatible with immunofluorescence analysis, relating to a cytochemical staining method compatible with immunofluorescence analysis and its applications. Chinese patent CN102043047B discloses a rapid and economical immunofluorescence method based on cell smears. However, no immunofluorescence staining methods for organoids with three-dimensional structures have been reported to date.

[0005] Traditional fluorescence immunoassay still primarily relies on the observation and interpretation of single tissue sections. The limitation of traditional single tissue sections is that they only reflect the condition of a single cross-section, which differs significantly from the three-dimensional morphology in living tissue, resulting in substantial information loss and difficulty in identifying the overall picture. To facilitate observation and research of tissues on three-dimensional models, some three-dimensional imaging techniques have emerged. For example, anatomical reconstruction based on CT or MRI is relatively mature; however, it can only observe the anatomical structure of organs and tissues, primarily serving clinical applications and unable to effectively reflect the cellular level within tissues. With the deepening of research in organoids and other biomaterials, exploring the spatial distribution relationships of various tissue cells and the spatial expression characteristics of various functional markers has become crucial for further understanding the development and pathophysiological changes of three-dimensional structures such as organoids. Therefore, establishing a multi-index, high-resolution three-dimensional tissue model has become a bottleneck that urgently needs to be overcome. Given the dual requirements of multi-index and high resolution in three-dimensional tissue models, this invention provides a microscope-based method for immunofluorescence staining and three-dimensional reconstruction of organoids. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing immunofluorescence staining techniques for three-dimensional structures such as organoids by providing a method for immunofluorescence staining and three-dimensional reconstruction of such structures.

[0007] In order to solve the above-mentioned technical problems, the first objective of this invention is to provide a method for immunofluorescence staining of organoids, comprising the following steps: collecting cultured organoids, taking the organoids under a magnification of 40 to 60 times or less, performing fixation, permeabilization and blocking treatment in sequence, then incubating with primary antibody solution and secondary antibody solution in sequence, followed by nucleus staining, and then mounting to obtain the mounted organoid.

[0008] Compared with the prior art, the method provided by the present invention has the following significant advantages: (1) The present invention avoids the tissue detachment problem that may be caused by the immunofluorescence staining method of climbing slides and paraffin sections for three-dimensional structures such as organoids, and the staining results are better. This method is very suitable for three-dimensional structures such as organoids; (2) The method provided by the present invention can maintain the structural integrity of three-dimensional structures such as organoids throughout the process, which is convenient for the reconstruction of three-dimensional structures and the accuracy of experimental results; (3) The present invention can select different numbers of organoids for staining according to the size and needs of organoids, which is conducive to sufficient staining and better staining effect, especially the cells inside the cell clusters are stained more fully; (4) The staining steps of the present invention are performed directly under magnification of 40 to 60 times (e.g., the stereomicroscope described below), which is simple to operate and only requires 1 drop of antibody solution each time, saving reagents; (5) The present invention can perform multiple combinations of staining on multiple organoids at one time, and can also perform multiple staining on one organoid.

[0009] Based on the above technical solution, the present invention can be further improved as follows.

[0010] The above-mentioned collection and culture of organoids can be carried out as follows: first, organoids are cultured, and when obvious growth of organoids is observed, the culture medium and Matrigel gel are blown away with a pipette tip coated with PBS buffer containing 0.1% BSA. The organoids are then enriched into centrifuge tubes coated with PBS buffer containing 0.1% BSA. The tubes are centrifuged at 400g for 3-7 minutes, and some of the Matrigel gel is deposited at the bottom of the centrifuge tube. The upper layer is obtained as an organoid Matrigel gel mixture.

[0011] Under the above conditions of magnification of 40 to 60 times or less, the organoids can be obtained as follows: the organoid matrigel mixture collected by centrifugation is resuspended in Organoid Harvest Solution, placed on a horizontal shaker at 4 degrees Celsius and mixed thoroughly for 40-50 minutes, centrifuged again at 400g centrifugation force for 3-7 minutes, and the organoids are obtained from the sediment at the bottom. Under the conditions of stereomicroscope magnification of 40 to 60 times, the organoids are accurately taken using a glass pipette with a tip diameter of about 500 μm.

[0012] Furthermore, the organoids are human cervical intraepithelial neoplasia organoids and / or human cervical cancer organoids.

[0013] The beneficial effects of adopting the above-mentioned further scheme are: the method provided by the present invention can perform multiple combinations of staining on multiple organoids at one time, and can also perform multiple staining on one organoid.

[0014] Furthermore, the fixation involves resuspending the obtained organoid in a fixative solution for fixation; the permeation involves transferring the fixed organoid into a permeabilizing solution for permeabilization at room temperature; and the sealing involves transferring the permeabilized organoid into a sealing solution for sealing.

[0015] Furthermore, the fixative is 4 wt% paraformaldehyde, 4 wt% neutral formaldehyde, or Zenker fixative; the permeabilizing solution is a PBS solution with a volume fraction of 0.1% Triton-X100, a PBS solution with a volume fraction of 0.1% Saponin, methanol, or acetone; and the blocking solution is a PBS solution with a mass content of 1% BSA or goat serum of the secondary antibody species.

[0016] Furthermore, the specific steps of fixation are as follows: under stereomicroscope magnification of 40 to 60 times, the organoid is taken, and the organoid is washed at least once with PBS buffer containing 1% BSA. Then, it is resuspended in the fixative and fixed at room temperature for more than 30 minutes.

[0017] The specific steps of the permeabilization are as follows: at least 3 drops of PBS solution with a mass content of 1% BSA are added to a glass dish to form at least 3 PBS droplets with 1% BSA. Under the condition of stereomicroscope magnification of 40 to 60 times, the fixed organoids are transferred into different PBS droplets with 1% BSA and washed at least 3 times. Then the organoids are transferred into the droplets of the permeabilization solution and permeabilized at room temperature for 0.5 to 2 hours.

[0018] The specific steps of the sealing process are as follows: under stereomicroscope magnification of 40 to 60 times, the organoids after permeation are transferred into droplets of the sealing solution and sealed at room temperature for 0.5 to 1.5 hours.

[0019] Furthermore, incubation with a primary antibody solution involves transferring the blocked organoids into the primary antibody solution for incubation; incubation with a secondary antibody solution involves transferring the organoids incubated with the primary antibody solution into the secondary antibody solution for incubation; and nucleostained organoids involve transferring the organoids incubated with the secondary antibody solution into a nucleostained solution for nucleostained organoids.

[0020] Furthermore, the primary antibody solution is prepared by dissolving an antigen solution (including P63 rabbit anti-human antigen solution and Krt13 mouse anti-human antigen solution, with a volume ratio of 1:1) in PBS with a mass ratio of 1:300; the secondary antibody solution is prepared by dissolving a diantigen solution (including FITC-labeled fluorescent diantigen solution and Cy3 goat anti-rabbit-labeled fluorescent diantigen solution, with a volume ratio of 1:1) in PBS with a mass ratio of 1:150; and the nucleoside solution is a DAPI solution.

[0021] Furthermore, the specific steps for incubation using the primary antibody solution are as follows: the primary antibody solution is dropped onto a glass dish to form at least one drop of primary antibody liquid. Under the condition of 40 to 60 times magnification of a stereomicroscope, the blocked organoid is transferred into the primary antibody liquid drop and incubated at 4°C for 12 to 24 hours.

[0022] The specific steps for incubation using the secondary antibody solution are as follows: under stereomicroscope magnification of 40 to 60 times, wash away the primary antibody from the non-specifically bound droplets of the primary antibody in the organoids incubated with the primary antibody solution, drop the secondary antibody solution onto a glass dish to form at least one droplet of secondary antibody, transfer the organoids into the droplet of secondary antibody, and incubate at room temperature for 1 to 2 hours;

[0023] The specific steps for nucleation are as follows: under stereomicroscope magnification of 40 to 60 times, wash away the secondary antibody in the organoid that has not specifically bound to the secondary antibody droplet after incubation with secondary antibody solution, drop the nucleation solution onto a glass dish to form at least one nucleation droplet, transfer the organoid into the nucleation droplet, and nucleate at room temperature for 5 to 10 minutes.

[0024] Furthermore, the mounting process involves adding the nucleated organoid onto a glass slide, followed by adding an anti-fluorescence quenching agent, and then surrounding the glass slide with a ring of silicone gel. After the silicone gel is at least semi-dry, a coverslip is placed on top to obtain the mounting of the organoid.

[0025] This method can create a three-dimensional space on the slide, avoiding compression of the organoids and allowing them to be detected in a spherical shape.

[0026] The detection and observation refers to observing the organoids after mounting using a confocal microscope.

[0027] The second objective is to provide a method for three-dimensional reconstruction of organoids obtained by immunofluorescence staining, comprising the following steps: capturing a fluorescence image of a different section of the organoid slide obtained by the immunofluorescence staining method described above for each vertical 1µm using the z-stack mode of a confocal microscope, obtaining at least 50 fluorescence images; and then reconstructing at least 503 fluorescence images in three dimensions using three-dimensional imaging software to obtain a three-dimensional reconstructed fluorescence image of the organoid.

[0028] The beneficial effects of adopting the above scheme are: the present invention can reconstruct three-dimensional structures such as organoids through three-dimensional imaging software, and clarify the internal structure of cell clusters, which is of great significance.

[0029] Furthermore, the 3D imaging software is Imaris 9.0.1. Attached Figure Description

[0030] Figure 1 This invention relates to a self-made glass pipette with a tip diameter of 500 μm;

[0031] Figure 2 Fluorescent images of cervical intraepithelial neoplasia organoids taken along the central axis for this invention: red: P63, green: KRT13, blue: DAPI, bar = 20um;

[0032] Figure 3 Fluorescent images of cervical cancer organoids taken along the central axis in this invention: red: P63, green: KRT13, blue: DAPI, bar = 20um;

[0033] Figure 4The image shows the three-dimensional structure of the cervical intraepithelial neoplasia organoid reconstructed using Imaris 9.0.1 software in this invention. Red: P63, Green: KRT13, Blue: DAPI.

[0034] Figure 5 The image shows the three-dimensional structure of cervical cancer organoids reconstructed using Imaris 9.0.1 software in this invention. Red: P63, Green: KRT13, Blue: DAPI. Detailed Implementation

[0035] The principles and features of this invention are described below. The examples given are for illustrative purposes only and are not intended to limit the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they should be performed according to the techniques or conditions described in the literature in this field, or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0036] Explanation of the source of experimental materials and reagents:

[0037] Organoids: Cervical intraepithelial neoplasia tissue and cervical cancer cells were enriched separately, resuspended in Matrigel gel, and seeded into plates. Culture medium containing special growth factors was added after 30 minutes.

[0038] Experimental reagents: 4% paraformaldehyde (Servicebio, Cat#G1101), 1% BSA (Servicebio, Cat#G5001), PBS (Servicebio), Triton X-100 (Solarbio, #T8200), primary antibody P63 rabbit anti-human (Abcam, Cat#124762), primary antibody Krt13 mouse anti-human (Abcam, Cat#92551, 1:150), fluorescent secondary antibody cy3 goat anti-rabbit (Servicebio, #GB21303), fluorescent secondary antibody FITC goat anti-mouse (Proteintech, #SA00003-1), DAPI (Servicebio, #G1012), anti-fluorescence quencher, silicone gel, glass pipettes with a 500µm tip (homemade, e.g.) Figure 1 ), Organoid harvestsolution (R&D, cat#1560173), Matrigel (Corning).

[0039] The aforementioned glass pipette with a tip diameter of 500 μm can be used to aspirate and transfer three-dimensional structures such as organoids between droplets. It consists of two parts: the glass pipette itself, with a tip diameter of 500 μm, and a rubber cap at the tip.

[0040] Equipment: low-speed centrifuge, low-temperature shaker, 4-degree refrigerator, confocal microscope (Zeiss LSM980 Airyscan2 inverted confocal microscope).

[0041] Software: Imaris 9.0.1.

[0042] Example

[0043] 1. Experimental Methods

[0044] 1.1 An immunofluorescence staining method for organoids

[0045] This embodiment relates to an immunofluorescence staining method for organoids, comprising the following steps: collecting cultured organoids, taking the organoids under a magnification of 40 to 60 times or less, performing fixation, permeabilization, and blocking treatments in sequence, then incubating with primary antibody solution and secondary antibody solution in sequence, followed by nucleus staining, and then mounting the organoids to obtain mounted slides for observation and detection.

[0046] Specifically, the steps include the following:

[0047] (1) Organoid culture and collection: Human cervical intraepithelial neoplasia cells or human cervical cancer cells were enriched, resuspended in Matrigel gel, and seeded into plates. After 30 minutes, a culture medium containing special growth factors was added. The culture medium for this organoid was: advanced DMEM / f12, 1X Hepes, 1-10X penicillin-streptomycin-amphoteric acid B mixed solution, 1X Glutamax, 1-2X mycoplasma removal reagent, 80-120 ng / ml Recombinant Human Noggin, 160-240 ng / ml FGF-7, 0-60 ng / ml EGF, 0.8-1.2 uM SB202190, 2-3 mM Nicotinamide, 1-1.5 mM N-acetylcysteine, 8-12 uM Forsklin, 1X B-27, 8-12 uM Y-27632, 400-600 nMA83-01.

[0048] After observing significant growth of organoids, the culture medium and Matrigel gel were agitated with a 1 ml pipette tip coated with 0.1% BSA and PBS buffer. The organoids were then enriched into 15 ml centrifuge tubes coated with 0.1% BSA and PBS buffer. The tubes were centrifuged at 400 g for 5 min, and the Matrigel gel precipitated to the bottom of the centrifuge tube. The upper layer was obtained as an organoid Matrigel gel mixture.

[0049] Note: During the organoid collection process, all consumables that come into contact with the Matrigel gel and organoids must be coated with 0.1% BSA in PBS buffer to prevent organoids from adhering to the surface of the consumables and causing unnecessary losses.

[0050] (2) Fixation: The organoid matrigel mixture collected by centrifugation in step (1) was resuspended in 5 ml of Organoid Harvest Solution, placed on a horizontal shaker at 4 degrees Celsius and mixed thoroughly for 45 min. It was then centrifuged again at 400 g for 5 min. The precipitate at the bottom was used to obtain the organoids. Under a stereomicroscope with a magnification of 40–60 times, a glass pipette with a tip diameter of approximately 500 μm (e.g., ...) was used. Figure 1 The organoids were taken, washed once with PBS buffer containing 1% BSA, resuspended, and fixed in 4% (w / w) paraformaldehyde at room temperature for at least 30 minutes.

[0051] (3) Membrane permeation: Add a few drops of 1% BSA PBS solution to a 6cm glass dish. Under the magnification of a stereomicroscope at 40-60x, use a glass pipette with a tip diameter of about 500µm (e.g., Figure 1 The fixed organoids were transferred into different PBS droplets containing 1% BSA and washed three times. This method of fluid exchange was named the droplet method (i.e., each step was performed under a stereomicroscope, using a glass pipette with a tip diameter of approximately 500 μm to transfer the three-dimensional structures, such as organoids, into different droplets). Subsequently, the organoids were transferred to a permeabilization solution, which consisted of 0.1% Triton-X100 (volume fraction) PBS solution, and permeabilized at room temperature for 1 hour.

[0052] (4) Blocking: Add a few drops of 1% BSA in PBS solution to a 6cm glass dish. Under the condition of stereomicroscope magnification of 40 to 60 times, use a glass pipette with a tip diameter of about 500um to transfer the permeabilized organoids into different drops of 1% BSA in PBS solution. Wash 3 times, and then transfer them into the blocking solution droplet to block at room temperature for 1 hour. The blocking solution is 1% BSA in PBS.

[0053] (5) Incubation of primary antibody: Dilute the primary antibody solution (including P63 antigen solution and Krt13 antigen solution with a volume ratio of 1:1) in PBS with 1% BSA at a volume ratio of 1:300 to obtain a primary antibody solution. Place the primary antibody solution on a 6cm glass dish to obtain a droplet of primary antibody. Add the cervical intraepithelial neoplasia organoid or cervical cancer organoid to the droplet using the droplet method under a stereomicroscope with a magnification of 40-60 times. Incubate overnight at 4°C in the dark, taking care to minimize the amount of liquid in the environment.

[0054] (6) Incubation of the second antibody: The second antigen solution (including FITC second antigen solution and Cy3 second antigen solution with a volume ratio of 1:1) was diluted in PBS with 1% BSA at a volume ratio of 1:150 to prepare the second antibody solution. After washing away the first antibody that did not bind to the target marker on the surface of the organoids in step (5) by the droplet method, each organoid was added to the droplets of the second antibody solution labeled with fluorescent groups and incubated at room temperature for 1 hour.

[0055] (7) Nucleus staining: Under the condition of stereomicroscope magnification of 40 to 60 times, after washing away the second antibody that did not bind to the first antibody on the surface of the organoids mentioned above by the droplet method, each organoid is placed in a DAPI droplet, and after 5 to 10 minutes, excess DAPI solution is washed away by the droplet method.

[0056] (8) Mounting: The nuclear-stained organoids are dropped onto a glass slide, followed by the addition of an anti-fluorescence quencher. A ring of silicone is then placed around the glass slide with a needle. After the silicone is semi-dry, a coverslip is placed on top to obtain the mounted organoids. This method can form a three-dimensional space on the glass slide, avoiding compression of the organoids and allowing them to be detected in a spherical shape.

[0057] (9) Observation and detection: The organoid cells after mounting were observed using a multi-channel confocal microscope.

[0058] 1.2 A method for three-dimensional reconstruction of organoids by immunofluorescence staining

[0059] This embodiment relates to a method for three-dimensional reconstruction of organoids by immunofluorescence staining, which includes the following steps:

[0060] (10) Three-dimensional imaging: Using the z-stack mode of a confocal microscope, one fluorescence image of the organoid was captured for each vertical 1µm. Then, the immunofluorescence images of at least 50 different sections of the organoid were reconstructed in three dimensions using Imaris software to obtain the three-dimensional reconstructed fluorescence images of the organoid.

[0061] 2. Experimental Results

[0062] Please refer to the attached image for fluorescence images. Figure 2-5 ,in Figure 2 Fluorescent images of cervical intraepithelial neoplasia organoids taken along the central axis according to the present invention: red: P63, green: KRT13, blue: DAPI, bar = 20um;

[0063] Figure 3 Fluorescent images of cervical cancer organoids taken along the central axis according to the present invention: red: P63, green: KRT13, blue: DAPI, bar = 20um;

[0064] Figure 4 The image shows the three-dimensional structure of the cervical intraepithelial neoplasia organoid reconstructed using Imaris 9.0.1 software according to the present invention. Red: P63, green: KRT13, blue: DAPI.

[0065] Figure 5 The image shows the three-dimensional structure of cervical cancer organoids reconstructed using Imaris 9.0.1 software according to the present invention. Red: P63, Green: KRT13, Blue: DAPI.

[0066] Depend on Figure 2-5 Confocal images and 3D reconstructed fluorescence images both show that the cervical basal cell marker P63 and the epithelial cell marker KRT13 are distributed in different locations on cervical intraepithelial neoplasia organoids and cervical cancer organoids. Furthermore, cervical intraepithelial neoplasia organoids differentiate from the outside in, while cervical cancer organoids consist of spheroids of cervical cancer tumor cells. P63 is only distributed in the outer layer of cervical intraepithelial neoplasia organoids, while it is distributed throughout the entire layer of cervical cancer organoid tumor cell spheroids. Immunofluorescence staining demonstrates that cervical intraepithelial neoplasia organoids have a different structure from cervical cancer organoids. In addition, cervical intraepithelial neoplasia organoids have a lower degree of malignancy compared to cervical cancer organoids.

[0067] In summary, the method provided by this invention has the following significant advantages: This invention avoids the tissue detachment problem that may occur with immunofluorescence staining methods using slides and paraffin sections for three-dimensional structures such as organoids, resulting in better staining results. This method is highly suitable for three-dimensional structures such as organoids. The method provided by this invention maintains the structural integrity of three-dimensional structures such as organoids throughout the process, facilitating the reconstruction of the three-dimensional structure and resulting in accurate experimental results. Selecting different numbers of organoids for staining according to their size and requirements is more conducive to thorough staining and better staining effects, especially for cells within cell clusters. The staining steps are performed directly under a stereomicroscope, simplifying the operation and requiring only one drop of diluted primary or secondary antibody solution each time, saving reagents and antibodies. Furthermore, it allows for multiple combinations of staining of multiple organoids at once, as well as multiple staining of a single organoid.

[0068] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for immunofluorescence staining of organoids, characterized in that, The process includes the following steps: collecting cultured organoids, taking the organoids at a magnification of 40 to 60 times, fixing, permeabilizing, and blocking them in sequence, then incubating them with primary antibody solution and secondary antibody solution in sequence, followed by nucleostained staining, and then mounting them to obtain mounted organoids. The organoids were collected using a glass pipette with a tip diameter of about 500 μm under a stereomicroscope magnification of 40 to 60 times. The fixation involves resuspending the obtained organoid in a fixative solution for fixation; the permeation involves transferring the fixed organoid into a permeabilizing solution for permeabilization at room temperature; and the sealing involves transferring the permeabilized organoid into a sealing solution for sealing. The specific steps of fixation are as follows: Under the condition of stereomicroscope magnification of 40 to 60 times, the organoid is taken, and the organoid is washed at least once with PBS buffer containing 1% BSA. Then it is resuspended in the fixative and fixed at room temperature for more than 30 minutes. The specific steps of the permeabilization are as follows: at least 3 drops of PBS solution with a mass content of 1% BSA are added to a glass dish to form at least 3 PBS droplets with 1% BSA. Under the condition of stereomicroscope magnification of 40 to 60 times, the fixed organoids are transferred into different PBS droplets with 1% BSA and washed at least 3 times. Then the organoids are transferred into droplets of permeabilization solution and permeabilized at room temperature for 0.5 to 2 hours. The specific steps of the sealing process are as follows: under stereomicroscope magnification of 40 to 60 times, the organoids after permeation are transferred into droplets of the sealing solution and sealed at room temperature for 0.5 to 1.5 hours. Incubation with primary antibody solution involves transferring the blocked organoids into the primary antibody solution for incubation; incubation with secondary antibody solution involves transferring the organoids incubated with primary antibody solution into the secondary antibody solution for incubation; nucleus staining involves transferring the organoids incubated with secondary antibody solution into a nucleus staining solution for nucleus staining. The specific steps for incubation using the primary antibody solution are as follows: the primary antibody solution is dropped onto a glass dish to form at least one drop of primary antibody. Under stereomicroscope magnification of 40 to 60 times, the blocked organoid is transferred into the primary antibody drop and incubated at 4°C for 12 to 24 hours. The specific steps for incubation using the secondary antibody solution are as follows: under stereomicroscope magnification of 40 to 60 times, wash away the primary antibody from the non-specifically bound droplets of the primary antibody in the organoids incubated with the primary antibody solution, drop the secondary antibody solution onto a glass dish to form at least one droplet of secondary antibody, transfer the organoids into the droplet of secondary antibody, and incubate at room temperature for 1 to 2 hours; The specific steps for nucleation are as follows: under stereomicroscope magnification of 40 to 60 times, wash away the secondary antibody in the organoid that has not specifically bound to the secondary antibody droplet after incubation with secondary antibody solution, drop the nucleation solution onto a glass dish to form at least one nucleation droplet, transfer the organoid into the nucleation droplet, and nucleate at room temperature for 5 to 10 minutes.

2. The method for immunofluorescence staining of organoids according to claim 1, characterized in that, The organoids are human cervical intraepithelial neoplasia organoids and / or human cervical cancer organoids.

3. The method for immunofluorescence staining of organoids according to claim 1, characterized in that, The fixative is 4 wt% paraformaldehyde, 4 wt% neutral formaldehyde, or Zenker fixative; the permeabilizing solution is 0.1% Triton-X100 PBS solution, 0.1% Saponin PBS solution, methanol, or acetone; and the blocking solution is 1% BSA PBS solution or goat serum of the secondary antibody species.

4. The method for immunofluorescence staining of organoids according to claim 1, characterized in that, The primary antibody solution was prepared by dissolving an antigen solution in PBS with a mass content of 1% BSA at a volume ratio of 1:300; the secondary antibody solution was prepared by dissolving two antigen solutions in PBS with a mass content of 1% BSA at a volume ratio of 1:150; and the nucleoside solution was a 2 μg / mLDAPI solution.

5. The method for immunofluorescence staining of organoids according to claim 1, characterized in that, The specific steps of the mounting are as follows: the nucleated organoid is dropped onto a glass slide, followed by the addition of an anti-fluorescence quencher, and a ring of silicone rubber is placed around the glass slide. After the silicone rubber is at least semi-dry, a coverslip is placed on the slide to obtain the mounted organoid.

6. A method for three-dimensional reconstruction of organoids by immunofluorescence staining, characterized in that, It includes the following steps: using the z-stack mode of a confocal microscope, capturing one fluorescence image per vertical 1 μm of different sections of the mounting slide of the organoid obtained by the immunofluorescence staining method of any one of claims 1 to 5, to obtain at least 50 fluorescence images; then using three-dimensional imaging software to perform three-dimensional reconstruction of the at least 50 fluorescence images to obtain three-dimensional reconstructed fluorescence images of the organoid.

Images