Organ-like microsphere embedding slicing method

Abstract

The invention relates to an organ-like microsphere embedding slicing method. The method specifically comprises the following steps: 1) preparing hydrogel microspheres and dyeing the hydrogel microspheres with fuchsin; (2) dehydrating the dyed hydrogel microspheres and the organoid together, then putting the dehydrated hydrogel microspheres and the organoid into an embedding mold, adding an embedding agent for embedding, and obtaining an embedded sample after the embedding agent is solidified; and 3) slicing the embedded sample, and receiving the sliced sample by using a glass slide. According to the method, the hydrogel microspheres are firstly dyed, and the dyed hydrogel microspheres and the organoid are embedded together, so that the peripheral area of the organoid can be obviously red, and the magenta dyeing area on the section sample can be clearly observed even after the section sample is sliced. The hydrogel microspheres dyed by fuchsin can be used as positioning marks of organoid-containing microspheres, can provide obvious prompts in the early transparent dehydration process and the later embedding section process, and effectively help an operator to judge the section repairing degree and the section collecting degree.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a method for embedding and slicing organoid microspheres. Background Technology

[0002] Organoids are artificially constructed three-dimensional cellular systems that possess structures and functions similar to real biological organs. The development and research in this field has become a hot topic in modern medicine and biology, promising to provide humans with more accurate and efficient means of biological research and medical treatment. When organoids are used for drug screening and disease model research, their size needs to be controlled within the range of 200–800 μm to ensure that the cells can obtain sufficient nutrients and oxygen, and that drugs can penetrate effectively.

[0003] Paraffin embedding is a commonly used tissue preparation method in histological and pathological studies. Its main purpose is to embed biological tissue samples that have been fixed, dehydrated, cleared, and impregnated with paraffin into blocks to facilitate subsequent sectioning and observation.

[0004] OCT (Optimal Cutting Temperature) embedding is a commonly used embedding method in histological studies, especially suitable for samples that need to maintain the integrity of enzyme activity, antigenicity, and nucleic acid in the tissue.

[0005] However, the objects commonly processed by various embedding techniques are all kinds of biological tissue samples, whose size ranges from millimeters to centimeters. When dealing with organoid samples (such as the small organoids mentioned above), the sample size is too small to be observed with the naked eye, and most organoid samples are semi-transparent, so it is difficult to determine the accurate location of the sample during the sectioning process, which affects the sectioning effect. Summary of the Invention

[0006] The purpose of this invention is to disclose a method for embedding and slicing hydrogel organoid microspheres, in order to solve one or more technical problems existing in the prior art and provide at least one beneficial option or create conditions.

[0007] This invention aims to provide a method for embedding and sectioning organoid microspheres. Specifically, it includes the following steps: 1) Prepare hydrogel microspheres and stain them with fuchsin; 2) The stained hydrogel microspheres and organoids were fixed and dehydrated together, then placed in an embedding mold, and embedding agent was added for embedding. After the embedding agent solidified, the embedded sample was obtained. 3) Slice the embedded sample and use a glass slide to hold the sliced ​​sample.

[0008] Because the hydrogel microspheres are stained beforehand and then embedded together with the organoids, the area around the organoids can show obvious red coloration, and even after slicing, the magenta-stained area on the sliced ​​sample can be clearly observed.

[0009] In the specific operation process, there are two options to choose from: (1) If the hydrogel microspheres containing organoids are directly stained with fuchsin, then in step 2), it is only necessary to place the hydrogel microspheres containing organoids in the embedding mold. The organoids in the hydrogel microspheres can be observed in the red area of ​​the final obtained slice sample.

[0010] (2) If there are concerns that fuchsin staining may have additional effects on the organoids within the hydrogel microspheres, hydrogel microspheres without organoid cells can be prepared separately and stained to serve as markers. In step 2), the hydrogel microspheres containing organoids and an appropriate amount of marker are placed together in an embedding mold, and the position of the marker is adjusted so that the hydrogel microspheres containing organoids are completely surrounded by the marker. The final obtained slice sample shows a transparent area within the red region, and the slice structure of the organoid is located within this transparent area.

[0011] According to a preferred embodiment of the present invention, the hydrogel microspheres in step 1) consist of a core and a shell. The core contains the organoid, and the shell is formed by cross-linking a photocrosslinked hydrogel material, encapsulating the core. Using hydrogel microspheres as a direct carrier to support and immobilize organoids effectively avoids sample loss and structural damage that occur during dehydration and clearing steps in conventional embedding methods. Furthermore, the hydrogel microspheres are visible to the naked eye, have a stable morphology, and are more convenient and controllable throughout the experimental process.

[0012] According to a preferred embodiment of the present invention, the photocrosslinked hydrogel material is selected from at least one of methacrylamide hyaluronic acid, methacrylamide polylysine, carboxymethyl chitosan, methacrylamide sodium alginate, or methacrylamide dextran. Depending on the different photocrosslinked hydrogel materials, the corresponding illumination conditions will vary, and may need to be replaced with blue light, ultraviolet light, etc.

[0013] According to a preferred embodiment of the present invention, the steps for preparing hydrogel microspheres containing organoids include: 1-1) using a mixture containing organoid cells as a core material, the photocrosslinked hydrogel material as a shell material, and corn oil containing 1% Span-80 as a flow carrier; 1-2) drawing the core material into a first syringe, drawing the shell material into a second syringe, drawing the flow carrier into a third syringe, loading the three syringes into the corresponding hopper of the droplet microfluidic printing device, performing 3D printing of hydrogel microspheres, and achieving crosslinking and molding of the printed product by ultraviolet light curing.

[0014] According to a preferred embodiment of the present invention, step 1) of staining involves adding fuchsin staining solution diluted with PBS to the hydrogel microspheres and staining at room temperature for 10 minutes.

[0015] According to a preferred embodiment of the present invention, the embedding is paraffin embedding, and the dehydration treatment includes the following steps: 2-1) soaking in 75% ethanol solution for 2 hours, centrifuging and discarding the supernatant; 2-2) soaking in 85% ethanol solution for 2 hours, centrifuging and discarding the supernatant; 2-3) soaking in 95% ethanol solution for 1 hour, centrifuging and discarding the supernatant; 2-4) soaking in anhydrous ethanol for 30 minutes, centrifuging and discarding the supernatant; 2-5) soaking in anhydrous ethanol for 30 minutes, centrifuging and discarding the supernatant; 2-6) soaking in benzene for 5-10 minutes, centrifuging and discarding the supernatant; 2-7) dimethyl... 2-8) Soak in benzene for 5-10 minutes, centrifuge and discard the supernatant; 2-9) Soak in xylene for 5-10 minutes, centrifuge and discard the supernatant; 2-10) Transfer to the embedding mold with a pipette, remove the residual liquid, wait for the liquid to evaporate and dry at room temperature, add paraffin wax melted at 65℃, soak in paraffin for 1 hour and then remove the pre-embedded block; 2-11) Add paraffin wax melted at 65℃ to cover the bottom of the embedding mold, place the pre-embedded block in the center of the embedding mold, wait for it to solidify slightly and then continue to add paraffin wax melted at 65℃ until the embedding mold is full, let stand for half a minute and then transfer to 4℃ to cool.

[0016] According to a preferred embodiment of the present invention, the embedding is OCT embedding, and the dehydration treatment includes the following steps: 2-1') washing the hydrogel microspheres with DPBS solution; 2-2') adding a 4% paraformaldehyde solution and reacting in a shaker at 4°C; 2-3') washing repeatedly with DPBS solution, adding a 30% sucrose solution, and dehydrating in a shaker at 4°C for 24 hours.

[0017] According to a preferred embodiment of the present invention, for the OCT embedding method, the embedding involves concentrating hydrogel microspheres at the center of the embedding mold, adding embedding agent, carefully concentrating the hydrogel microspheres into the middle layer of the embedding agent using a pipette tip, and then transferring the embedding mold to a -20°C environment for freezing.

[0018] According to a preferred embodiment of the present invention, the organoid microsphere embedding and sectioning method further includes the following steps: 4) Perform immunofluorescence staining on the sliced ​​samples.

[0019] According to a preferred embodiment of the present invention, for the OCT embedding method, step 4) of immunofluorescence staining specifically includes: 4-1) warming the slide containing the section sample; 4-2) washing with PBS solution to remove the OCT embedding agent; 4-3) adding blocking and permeabilizing solution to the section sample and incubating at room temperature for 1-2 hours; 4-4) aspirating the blocking and permeabilizing solution from the edge of the section sample, then adding an appropriate amount of primary antibody diluent and incubating overnight at 4°C; 4-5) aspirating the primary antibody diluent from the edge of the section sample, washing three times with PBS solution for 5 minutes each time; 4-6) adding secondary antibody diluent and DAPI staining solution to the section sample and incubating at room temperature in the dark for 2 hours; 4-7) aspirating the secondary antibody mixture from the edge of the section sample, washing three times with PBS solution for 5 minutes each time; 4-8) aspirating the liquid in the section sample, adding anti-fluorescence quencher, and sealing with a coverslip.

[0020] Compared with existing detection technologies, the present invention has the following advantages: Fuchsin-stained hydrogel microspheres can serve as localization markers for organoid microspheres, providing clear indications during the initial clearing and dehydration process and the subsequent embedding and sectioning process, effectively helping operators judge the degree of trimming and slide collection. Attached Figure Description

[0021] Figure 1 This is a photograph of the organoid-containing hydrogel microspheres prepared in Example 1; Figure 2 This is a photograph of the marker prepared in Example 2; Figure 3 These are photographs taken during the paraffin embedding and sectioning process in Example 3; Figure 4 These are photographs taken during the OCT embedding and slicing process in Example 4; Figure 5 These are H&E staining images of colorectal cancer organoids and breast cancer organoids from Example 5; Figure 6 This is an immunofluorescence result image of colorectal cancer organoids from Example 6; Figure 7 This is the immunofluorescence result of breast cancer organoids in Example 7. Detailed Implementation

[0022] The following embodiments further illustrate the content of the present invention, but should not be construed as limiting the present invention. Any modifications and substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the present invention are within the scope of the present invention.

[0023] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0024] Unless otherwise specified, the molecular biology experimental methods described in the following examples were performed in accordance with Molecular Cloning: A Laboratory Manual (3rd Edition) or the kit and product instructions. Unless otherwise specified, the biological materials used in the kits are commercially available.

[0025] Example 1: Preparation of organoid-containing hydrogel microspheres Based on a droplet microfluidic system, colorectal cancer organoids (containing 40% colorectal cancer organoid culture medium and 60% matrix gel) at concentrations of 40,000 / μL and 80,000 / μL were used as core materials, and HAMA containing 0.5% LAP was used as the outer shell material. These were transferred to 1 mL syringes, and corn oil containing 1% Span-80 was drawn into 50 mL syringes. The three syringes were then loaded onto the corresponding core, outer shell, and oil phase hoppers of the droplet microfluidic printing device for 3D printing of hydrogel organoid microspheres. The printed products were cross-linked and formed using UV curing. After 7 days of culture, single cells were observed to self-assemble into organoid clusters, and significant proliferation was observed (e.g., ...). Figure 1 (As shown).

[0026] Example 2: Preparation of the marker Following Example 1 above, the core material was replaced with PBS solution, and the prepared hydrogel microspheres were used for fuchsin staining. After washing the prepared hydrogel microspheres, 50 μL of fuchsin staining solution was added, and staining was performed at room temperature for 10 minutes. The hydrogel microspheres were visibly red to the naked eye. Microscopic observation of the marker showed that the fuchsin staining penetrated the entire microsphere, exhibiting good color development and localization effects (e.g., ...). Figure 2 (As shown).

[0027] Example 3: Paraffin Embedding Experiment 1) Prepare organoid-containing hydrogel microspheres according to Example 1, and stain the hydrogel microspheres with fuchsin according to the method described in Example 2.

[0028] 2) Resuspend the stained hydrogel microspheres obtained in step 1) with 1 mL of DPBS, then transfer them to a 1.5 mL EP tube for washing, centrifuge at 600 rpm for 1 minute to concentrate the microspheres at the bottom of the tube, and discard the supernatant.

[0029] 3) Dehydration and clearing: The stained hydrogel microspheres were soaked in 75% alcohol solution for 2 hours, 85% alcohol solution for 2 hours, 90% alcohol solution for 2 hours, 95% alcohol solution for 1 hour, anhydrous ethanol for 30 minutes, and then replaced with new anhydrous ethanol for another 30 minutes. Then, they were soaked in benzene for 5-10 minutes and xylene for 5-10 minutes, and then replaced with new xylene for another 5-10 minutes. After each soaking step, the microspheres were centrifuged at 600 rpm for 1 minute to concentrate the stained hydrogel microspheres at the bottom of the tube, and the supernatant was discarded.

[0030] 4) Wax impregnation: The stained hydrogel microspheres are placed in the embedding mold, and molten paraffin at 65°C is added for 1 hour. After removal, the pre-embedded block is obtained.

[0031] 5) Embedding: First, add a small amount of 65°C melted paraffin wax to the embedding mold to cover the bottom of the embedding mold. Carefully pick up the pre-embedded block and drip it into the center of the embedding mold. After it solidifies slightly, continue to add 65°C melted paraffin wax until the embedding mold is filled. Let it stand for half a minute and then transfer it to 4°C overnight.

[0032] 6) Sectioning: Remove the embedded sample from the embedding cassette and place it on a paraffin microtome. First, roughly trim the wax block. Once the red sample is cut out, quickly adjust the thickness and begin slicing. The slice thickness should be 3-5 μm. Use a brush to pull the cut wax slice outwards, and use tweezers to place the wax slice into a spreader (around 42°C) to allow it to spread.

[0033] 7) Retrieving slides: Use a glass slide to retrieve tissue paraffin slides and adhere the tissue onto the glass slide; retrieve two tissue paraffin slides from each glass slide.

[0034] 8) Drying the slides: Place the retrieved slides on a slide dryer at 42-45℃, and then transfer them to a 37℃ oven to dry overnight. If tissue staining cannot be performed in time, store them in a refrigerator at 4℃.

[0035] Part of the process for preparing paraffin-embedded sections is as follows: Figure 3 As shown, obvious red dots can be clearly observed in the paraffin infiltration and sectioning steps, which are the stained hydrogel microspheres with organoids.

[0036] Example 4: Preparation of OCT embedded sections of colorectal cancer organoids and breast cancer organoids Preparation process: 1) Prepare organoid-containing hydrogel microspheres and markers according to Example 1 and Example 2, respectively.

[0037] 2) Resuspend the hydrogel microspheres in 1 mL DPBS, then transfer them to a 1.5 mL EP tube for washing. Centrifuge at 600 rpm for 1 minute to concentrate the microspheres at the bottom of the tube, and discard the supernatant.

[0038] 3) Add 1 mL of 4% paraformaldehyde solution and place it on a shaker in a 4°C refrigerator overnight for fixation.

[0039] 4) Remove the paraformaldehyde, wash repeatedly with DPBS, and then remove the DPBS; 5) Add 1 mL of 30% sucrose solution (30 mg sucrose, add 70 mL ddH2O, stir well, store at 4℃ for later use), place on a shaker in a 4℃ refrigerator, and dehydrate for 24 hours.

[0040] 6) Carefully aspirate the sucrose solution until about 30-50 μL of liquid remains at the bottom, then transfer the bottom liquid to the embedding mold.

[0041] 7) Carefully aspirate excess solution with a 10 μL pipette, gather the organoid-containing hydrogel microspheres into the center of the mold, and let them stand to dry for 10-15 minutes.

[0042] 8) Add 20 μL of the marker to the mold to be embedded, carefully remove the excess solution with a 10 μL pipette, gather the marker next to the organoid microspheres, and let it stand to dry for 10-15 minutes.

[0043] 9) After the two types of microspheres have dried, mix them thoroughly and concentrate them in the center of the embedding mold. Add OCT embedding agent for embedding. Carefully concentrate the hydrogel microspheres into the middle layer of the embedding agent using a 10 μL pipette, and then transfer them to a -20℃ refrigerator for storage.

[0044] 10) Set the cryostat temperature to -20℃, install the slicing blade, and place the embedded sample block inside the cryostat to equilibrate the temperature.

[0045] 11) Remove the sample block from the embedding mold and fix it onto the sample holder after freezing with a small amount of OCT embedding agent.

[0046] 12) Use the fast forward button to move the sample close to the blade, and use the slow forward button to start trimming the block. Observe whether the red marker is cut, and adjust the plane to be cut.

[0047] 13) Once the section reaches the marker, adjust the anti-roll plate, set the section thickness to 5~10 μm, rotate the microtome wheel, and begin collecting the section. The section should be complete and smooth.

[0048] 14) Place the glass slide close to the cut section and adhere the tissue to the glass slide; generally, 3 to 5 sections of sample are attached to one glass slide.

[0049] 15) Allow the slide to warm to room temperature for 30 minutes. Carefully add PBS solution to the sample slide from the edge of the slide using a Pasteur pipette. Wash repeatedly until the OCT embedding agent is completely dissolved and cleaned.

[0050] 16) Draw a circle around the section sample on the slide using an immunohistochemical PAP pen, add an appropriate amount of blocking permeation solution (PBS + 5% serum of the corresponding secondary antibody species + 0.3% Triton X-100) into the circle, place the slide in a humidified chamber, and let it stand at room temperature for 1 hour.

[0051] 17) Use absorbent paper to blot away the blocking permeate from the edge of the sample ring, then add an appropriate amount of primary antibody dilution buffer (PBS + 1% BSA + 0.3% Triton X-100) to dilute the primary antibody according to the ratio. Place the slide in a humidified chamber and incubate overnight at 4°C.

[0052] 18) Use absorbent paper to blot away the primary antibody dilution solution from the edge of the sample ring, and wash with PBS solution 3 times, 5 minutes each time.

[0053] 19) Dilute the secondary antibody and DAPI staining solution of the corresponding species with PBS in proportion, add an appropriate amount of secondary antibody and DAPI mixture, and incubate in a humidified chamber at room temperature in the dark for 2 hours.

[0054] 20) Use absorbent paper to wipe away the secondary antibody mixture from the edge of the sample ring, and wash with PBS staining solution 3 times, 5 minutes each time.

[0055] 21) Use absorbent paper to wipe away the liquid in the sample ring, add an appropriate amount of anti-fluorescence quenching agent while the sample is still wet, cover with a coverslip, and take pictures for detection using a fluorescence microscope.

[0056] During the slicing process, the areas marked by red markers can be clearly seen (e.g., Figure 4 (As shown). H&E staining results are as follows: Figure 5 As shown in the figure (20×), the organoids of colorectal cancer are structurally intact, some of which are solid clumps and some contain cystic cavities; while the organoids of breast cancer are solid clumps.

[0057] Example 5: Immunofluorescence staining test Immunofluorescence staining was performed on OCT-embedded sections of colorectal cancer organoids, and the results are as follows: Figure 6 As shown in the figure. Blue represents cell nuclei stained with DAPI, red represents CDX2 staining, and green represents CD20 staining. The results show that both CDX2 and CD20 are positively expressed and located on the cell membrane (20×). Immunofluorescence staining was performed on OCT-embedded sections of breast cancer organoids, and the results are shown below. Figure 7 As shown in the figure. Blue represents the cell nuclei after DAPI staining, and HER2 and PR staining (both red, located in the cytoplasm) are both positive (400×).

[0058] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

Claims

1. A method for embedding and sectioning organoid microspheres, characterized in that, Including the following steps: 1) Prepare hydrogel microspheres and stain them with fuchsin; 2) The stained hydrogel microspheres and organoids were fixed and dehydrated together, then placed in an embedding mold, and embedding agent was added for embedding. After the embedding agent solidified, the embedded sample was obtained. 3) Slice the embedded sample and use a glass slide to hold the sliced ​​sample.

2. The method for embedding and slicing organoid microspheres according to claim 1, characterized in that, Step 1) The hydrogel microspheres consist of a core and a shell. The core contains the organoids, and the shell is formed by cross-linking a photocrosslinked hydrogel material, which encapsulates the core.

3. The method for embedding and slicing organoid microspheres according to claim 2, characterized in that, The photocrosslinked hydrogel material is selected from at least one of methacrylamide hyaluronic acid, methacrylamide polylysine, carboxymethyl chitosan, methacrylamide sodium alginate, or methacrylamide dextran.

4. The method for embedding and sectioning organoid microspheres according to claim 2 or 3, characterized in that, The steps for preparing the hydrogel microspheres include: 1-1) A mixture containing organoid cells is used as the core material, the photocrosslinked hydrogel material is used as the shell material, and corn oil containing 1% Span-80 is used as the flow carrier; 1-2) The core material is drawn into the first syringe, the outer shell material is drawn into the second syringe, and the fluid carrier is drawn into the third syringe. The three syringes are loaded into the corresponding hoppers of the droplet microfluidic printing device to perform 3D printing of hydrogel microspheres, and the printed products are cross-linked and formed by ultraviolet light curing.

5. The method for embedding and sectioning organoid microspheres according to claim 1, characterized in that, Step 1) The staining involves adding fuchsin staining solution diluted with PBS to the hydrogel microspheres and staining at room temperature for 10 minutes.

6. The method for embedding and sectioning organoid microspheres according to claim 1, characterized in that, The embedding is paraffin embedding, and includes the following steps: 2-1) Soak in 75% ethanol solution for 2 hours, then centrifuge and discard the supernatant; 2-2) Soak in 85% ethanol solution for 2 hours, then centrifuge and discard the supernatant; 2-3) Soak in 95% ethanol solution for 1 hour, then centrifuge and discard the supernatant; 2-4) Soak in anhydrous ethanol for 30 minutes, centrifuge and discard the supernatant; 2-5) Soak in anhydrous ethanol for 30 minutes, centrifuge, and discard the supernatant; 2-6) Soak in alcohol and benzene for 5-10 minutes, then centrifuge and discard the supernatant; 2-7) Soak in xylene for 5-10 minutes, centrifuge and discard the supernatant; 2-8) Soak in xylene for 5-10 minutes, centrifuge and discard the supernatant; 2-9) Transfer the liquid to the embedding mold using a pipette, remove the remaining liquid, wait for the liquid to evaporate and dry at room temperature, add paraffin wax melted at 65℃, and remove the pre-embedded block after 1 hour of wax impregnation; 2-10) Add 65℃ melted paraffin wax to cover the bottom of the embedding mold, place the pre-embedded block in the center of the embedding mold, and continue to add 65℃ melted paraffin wax until the embedding mold is full after it has solidified slightly. Let it stand for half a minute and then transfer it to 4℃ to cool.

7. The method for embedding and sectioning organoid microspheres according to claim 1, characterized in that, The embedding is OCT embedding, which includes the following steps: 2-1') Wash the hydrogel microspheres with DPBS solution; 2-2') Add a 4% paraformaldehyde solution and react in a shaker at 4°C; 2-3') Wash repeatedly with DPBS solution, add 30% sucrose solution, and dehydrate on a shaker at 4°C for 24 hours.

8. The method for embedding and sectioning organoid microspheres according to claim 7, characterized in that, Step 2) involves placing multiple hydrogel microspheres around and stacking them around the organoid.

9. The method for embedding and sectioning organoid microspheres according to claim 8, characterized in that, It also includes the following steps: 4) Perform immunofluorescence staining on the sliced ​​samples.

10. The method for embedding and sectioning organoid microspheres according to claim 9, characterized in that, Step 4) The immunofluorescence staining specifically includes: 4-1) Warm the glass slide containing the sliced ​​sample back to room temperature; 4-2) Wash with PBS solution to remove the embedding agent; 4-3) Add the blocking and permeation solution to the sliced ​​sample and let it stand at room temperature for 1-2 hours; 4-4) Remove the blocking and permeation solution from the edge of the sliced ​​sample, then add an appropriate amount of primary antibody dilution solution and incubate overnight at 4°C; 4-5) Aspirate the primary antibody dilution solution from the edge of the sliced ​​sample, and wash with PBS solution 3 times, 5 minutes each time; 4-6) Add secondary antibody dilution solution and DAPI staining solution to the sliced ​​sample and incubate at room temperature in the dark for 2 hours; 4-7) Aspirate the secondary antibody mixture from the edge of the sliced ​​sample, and wash three times with PBS solution, each time for 5 minutes; 4-8) Absorb the liquid from the sliced ​​sample, add an anti-fluorescence quencher, and cover with a coverslip.

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