An automated resin slicing method without tabletting

Abstract

The present invention discloses an automated resin slicing method without tablet pressing, and relates to the technical field of tissue slicing. The resin slicing method comprises the steps of preparing resin embedded blocks, trimming blocks, slicing, pretreatment of tablet spreading, tablet spreading, scooping, baking, staining, and sealing, wherein, in S3 slicing, tablet pressing is not performed, and a slicer is used to quickly cut a large number of slices of required thickness; and severely curled slices are collected. The advantages are: tablet pressing is not required during slicing, and the difficulty of operation is greatly reduced; compared with the traditional manual slicing method, the slicing speed of the method of the present invention is significantly improved, and a large number of high-quality slices can be obtained within 5 minutes, while it is extremely difficult and requires operation to obtain a high-quality slice using the general tablet pressing slicing method.

Description

Technical Field

[0001] The invention relates to the technical field of microscopic tissue slicing, in particular to an automatic resin slicing method without tablet pressing. Background Art

[0002] Resin semi-thin sectioning technology, with its unique thickness between ultra-thin sections and paraffin sections, has become a common method in the field of microscopic observation. This technology uses resin as an embedding agent, and after fine treatments such as fixation, dehydration, and infiltration, it can prepare microscopic tissue sections with high clarity and high resolution. Compared with paraffin sections, it avoids the problem of overlapping cell components, allowing researchers to gain a more detailed insight into the internal structure and function of cells. In addition, resin semi-thin sectioning technology is not only suitable for basic scientific research such as cytology and embryology, but also shows broad application prospects in medical fields such as pathological diagnosis.

[0003] In the process of resin semi-thin sectioning, using a microtome to section is the most difficult step. Since the resin material itself has a certain toughness and elasticity, the section is easily curled due to external force squeezing or stretching during the sectioning process. Curled sections tend to fold together when exposed to water and cannot be fully unfolded, making it difficult to observe under a microscope.

[0004] Although some current slicers are equipped with automatic slicing functions, in order to avoid curling of slices, manual slicing and the use of corresponding tools to press slices are required. However, this often requires long-term practice and experience accumulation to master stable slicing techniques. For the processing of a large number of samples, manual slicing may become a time-consuming and tedious task. In addition, long-term operation may also place a certain burden on the operator's physical strength and energy. Therefore, improving relevant technical means to achieve automated slicing has become an urgent problem to be solved in achieving high-throughput slicing. Summary of the invention

[0005] The purpose of the present invention is to propose a method suitable for automatic resin slicing without tablet pressing in view of the background technology. In the method, manual slicing and tablet pressing are not required, and a machine is used for automatic slicing. Then, a large number of severely curled slices obtained by automatic slicing are collected, transferred to a treatment liquid for pretreatment, and the resin slices are unfolded and fully unfolded by means of a water tank. Since tablet pressing is not required, slicing is very easy to use and can be mastered after a short learning period.

[0006] The technical solutions adopted are as follows:

[0007] The present invention provides a method for preparing resin slices of wheat grains at maturity, comprising the following steps:

[0008] S1, preparing resin embedded blocks;

[0009] S2, block trimming: Trim the resin embedded block prepared in S1, remove the excess resin around it, and trim a complete tissue block section;

[0010] S3, Slicing: Without pressing, use a slicer to quickly cut a large number of slices of the required thickness; collect severely curled slices;

[0011] S4, pretreatment of the slide: prepare a treatment solution according to the ratio of saturated potassium hydroxide anhydrous ethanol solution and acetone in 2:1, drop a small amount of the treatment solution onto the slide, and transfer the collected severely curled sections onto the slide;

[0012] S5. Spread the slide: Insert the slide diagonally into the water tank quickly. The slice immersed in water will quickly float up and spread out.

[0013] S6. Pick up the slices: Use a new slide to pick up the fully unfolded slices;

[0014] S7, drying: transfer the glass slide in S6 to a 60°C oven for drying;

[0015] S8, staining: stain the slices in S7 with a staining solution, and then transfer to a 60°C oven for drying after staining;

[0016] S9, sealing: Use sealing medium to seal the slices in S8.

[0017] In a further preferred embodiment of the technical solution of the present invention, in S1, the preparation of the resin embedded block is carried out according to the experimental requirements, including but not limited to plant roots, stems or seeds; specifically, a tissue block of a certain thickness is cut, and the preparation of the embedded block is completed according to the resin slice preparation process of fixation, dehydration and infiltration. In S1 of the present invention, the time for each step of the preparation of the resin embedded block depends on the material conditions, and the relevant general production method can be used. The container selection during embedding should be appropriate, and there should not be too much resin blank.

[0018] In a further preferred embodiment of the technical solution of the present invention, in S2, the block can be trimmed manually or automatically by a machine until a complete cross section of the tissue block is obtained.

[0019] In a further preferred embodiment of the technical solution of the present invention, the slicing method in S3 is as follows:

[0020] S31. Install a tungsten steel knife on the slicer and clamp it in the knife clamp;

[0021] S32, adjusting the slice to a desired thickness, and adjusting the position of the blade and the resin embedding block so that the blade edge is aligned with the resin embedding block;

[0022] S33, adjusting the slicer to automatically slice, adjusting the slicing speed, retracting the blade after slicing for a period of time, and clamping the severely curled slices that are stuck, and clamping them together for transfer.

[0023] In a further preferred embodiment of the technical solution of the present invention, in S33, the rotating arm on the slicer can be manually rotated to speed up the slicing speed. In S33 of the present invention, if the automatic slicing speed is slow, the rotating arm can be quickly rotated with the right hand to cut 20-40 slices, which takes about 10 seconds, and then the severely curled slices are collected on a glass slide with a treatment solution.

[0024] Further preferred embodiment of the technical solution of the present invention is that in S4, the preparation method of the treatment liquid is as follows: add excess potassium hydroxide to anhydrous ethanol, seal with plastic wrap to prevent water from entering, heat in a 60°C water bath until the solution turns yellow, absorb 20 ml of the supernatant into a test tube, and mix with acetone in a ratio of 2:1; a light yellow treatment liquid is obtained.

[0025] In S4 of the present invention, the treatment liquid is stored in a refrigerator at 4°C and can generally be stored for 2-3 days; if the storage time is too long, the treatment liquid will gradually turn brown and can continue to be used, but will produce a pungent odor.

[0026] In a further preferred embodiment of the technical solution of the present invention, in S4, 200ul of the treatment solution is sucked up by a pipette and added to the glass slide, the treatment solution will spread rapidly, multiple slices are clamped with tweezers and placed on the glass slide so that each slice is in contact with the treatment solution, and then the glass slide is quickly and completely immersed in a 60°C water tank. The slices immersed in the water float up rapidly and relax from being severely curled in the process.

[0027] In a further optimization of the technical solution of the present invention, after the slices transferred in S6 float to the water surface, not all of them are intact, and it is necessary to pick the slices that look good to the naked eye, use a brush to gently move the slices so that the slices are partially in contact with the slide, and slowly lift the slide off the water surface.

[0028] In S6 of the present invention, it is generally preferred that 1 / 3 to 1 / 2 of the slices be transferred.

[0029] In a further preferred embodiment of the technical solution of the present invention, the drying time in S7 and S8 is longer than 2 hours. If the drying time is too short, the slices are easy to fold during dyeing. The drying time is longer than 2 hours, so that the slices treated with the treatment liquid will not overlap if the drying time is sufficient.

[0030] In a further preferred embodiment of the technical solution of the present invention, a sealing agent is used in S9, a drop of which is dropped on the slice, and then covered with a coverslip and squeezed, and the sealing agent will gradually spread to the entire slice.

[0031] In S9 of the present invention, the flattened slice is placed on a glass slide, and a 1 ml pipette is used to add the sealing agent. After cutting off 1 cm of the pipette head, the sealing agent is aspirated slowly to avoid bubbles, and then dripped onto the slice; finally, a cover glass is placed and squeezed to allow the sealing agent to gradually diffuse to the entire slice, thereby completing the slice sealing.

[0032] The method of the present invention is suitable for automatic slicing, and adopts the steps of slicing pretreatment and water tank slicing to enable folded and severely curled slicing to be normally unfolded, thereby achieving the elimination of the need for slicing and automatic slicing, reducing the difficulty of slicing production, realizing batch resin slicing, and greatly improving slicing efficiency.

[0033] The method of the present invention solves the problem of manual reliance, time-consuming and labor-intensive process in the resin slicing process, and can obtain multiple high-quality slices at one time, so that the slice tissue and cell structure are complete, clear and contrasting, thus providing technical support for the production of resin slices.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] 1. The method of the present invention does not require tableting during slicing, and the operation difficulty is greatly reduced.

[0036] 2. The slicing speed of the method of the present invention is significantly improved. For a skilled operator, it may take 10-20 minutes to cut a complete slice with traditional manual slicing, and the time cost is even higher for a novice. The method of slicing can obtain more than 10 complete slices within 5 minutes, and the slicing efficiency is significantly improved.

[0037] 3. The method of the present invention uses special treatment liquid pretreatment and water tank slice unfolding to enable folded and severely curled slices that are generally considered waste slices to be unfolded normally, and the slice quality is improved compared to general slices.

[0038] 4. The method of the present invention is applicable to the automatic slicing function of the slicer, and even manual slicing can significantly improve the slicing efficiency. Specifically, in traditional slicing, the slice needs to be pressed by hand to prevent curling, and once the slice is curled, it is difficult to fully unfold it; however, the method for preparing slices has no requirements on the curling of the slices, and even if the slices are curled, they can be fully unfolded, so there is no need to press, saving a lot of time.

[0039] 5. The method of the present invention is widely applicable to the common problem of pressing resin slices, and can be applied to a variety of tissues.

[0040] 6. Compared with the traditional manual slicing method, the slicing speed of the method of the present invention is significantly improved, and a large number of high-quality slices can be obtained within 5 minutes, while it is extremely difficult and requires operation to obtain a high-quality slice by the general pressed slice method. The present invention solves the problem of relying on manual labor, time-consuming and labor-intensive in the resin slicing process, and can cut multiple high-quality slices at one time, making the slices with complete tissue and cell structure, clear and obvious contrast, providing technical support for the production of resin slices. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 The slice sample group a is prepared by the fully automatic slicer without tabletting according to the method of the present invention, and 3 slices are selected from the sample group a, and the sample numbers are 1, 2 and 3 respectively;

[0042] Figure 2 The slice sample group b is made by manual slices of traditional resin slices. Three slices are selected from the samples in group b, and the sample numbers are 1, 2 and 3 respectively;

[0043] Figure 3 Yes Figure 1 The slice sample group c after using image J to identify the protein staining area in the middle slice;

[0044] Figure 4 Yes Figure 2 The slices in the middle section were sample group d after using Image J to identify the protein staining area. DETAILED DESCRIPTION

[0045] The technical solution of the present invention is described in detail below, but the protection scope of the present invention is not limited to the embodiments.

[0046] To make the content of the present invention more clearly understood, the following Figure 1-Figure 4 The specific implementation manner is further described.

[0047] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0048] This embodiment is a method suitable for automated resin slicing without tabletting, comprising the following steps:

[0049] S1. Preparation of resin embedded blocks:

[0050] ① Pick 2-4 normal-shaped wheat, rice and corn grains respectively, soak them in water to soften them in advance; use a razor blade to cut a 2-3 mm thick tissue block from the middle of the grain. When cutting the tissue block, do not squeeze the wheat grain hard to avoid deformation of the tissue due to squeezing;

[0051] ② Prepare 10% paraformaldehyde-2.5% glutaraldehyde fixative: 250 mL 0.2 mol / L phosphate buffer (pH 7.4) + 100 mL 10% paraformaldehyde aqueous solution + 50 mL 25% glutaraldehyde aqueous solution;

[0052] ③ Use forceps to grab the tissue block and place it in a 2 ml centrifuge tube. Use a pipette to add 1-1.5 ml of fixative solution and soak it in the fixative solution for 48 hours at room temperature to fully fix it.

[0053] ④ Use a pipette to draw 1.5 ml of 10× PBS buffer into the centrifuge tube to fully wash away the fixative. This step takes 20 minutes each time and is repeated 6-8 times.

[0054] ⑤ Use a step-by-step dehydration method to prepare 25%, 50%, and 75% ethanol aqueous solutions: the ratio of anhydrous ethanol to water is 1:3, 1:1, and 3:1, and the tissue blocks are dehydrated step by step according to the gradient of 25%, 50%, 75%, 100%, and 100%, each level for 10 min; in this step, pay attention to the ethanol volatile nature, and immediately add a new gradient ethanol aqueous solution after the original gradient ethanol aqueous solution is absorbed cleanly;

[0055] ⑥Prepare London white glue resin: Wash the magnetic stirrer, beaker, and measuring cylinder glass rod in advance and put them in an oven to dry. Prepare London white glue resin according to the ratio of 40 ml resin + 0.8 g catalyst, stir it magnetically to fully dissolve it, and place it in a 4℃ refrigerator for 24 hours before use (the prepared resin will turn yellow if stored in the refrigerator for too long, which is a normal phenomenon);

[0056] ⑦ Prepare resin ethanol solutions with concentrations of 20%, 40%, 60%, and 80%, with the ratio of resin to anhydrous ethanol being 1:4, 2:3, 3:2, and 4:1;

[0057] ⑧Permeabilize the tissue block step by step according to the gradient of 20%, 40%, 60%, 80%, 100%, and 100%; each step takes 2 hours;

[0058] ⑨ Place the infiltrated tissue block with the cross section facing downward in the embedding mold (here, select No. 1 transparent capsule for wheat grains), use a pipette to slowly inject resin until the tissue block is immersed, and prevent bubbles from being generated during the process. After the resin is injected, use small tweezers to place the tissue block to prevent it from tilting;

[0059] ⑩ Place the embedding mold in a 60°C oven for 1-2 days to harden the resin (the temperature must be set to 60°C and the polymerization time cannot exceed 2 days, otherwise it will be difficult to slice), and obtain a resin-embedded block.

[0060] S2, repair block:

[0061] The block can be trimmed manually or automatically by a machine to a complete cross section of the tissue block. Manual or automatic modification is known to those skilled in the art.

[0062] S3, Slice:

[0063] The slicing method is as follows:

[0064] S31. Install a tungsten steel knife on the slicer and clamp it in the knife clamp;

[0065] S32, adjusting the slice to a desired thickness, and adjusting the position of the blade and the resin embedding block so that the blade edge is aligned with the resin embedding block;

[0066] S33, adjust the slicer to slice automatically, adjust the slicing speed, retract the blade after slicing for a while, and clamp the severely curled slices that are stuck on the slices, and clamp them together for transfer. If you feel that the automatic slicing speed is slow, you can also quickly rotate the rotating arm with your right hand to cut 20-40 slices, which takes about 10 seconds.

[0067] In this embodiment, in the slicing step of S3, the slices were placed in a 60° C. oven for 12 h to prevent detachment during the staining process.

[0068] S4. Pre-processing of the exhibition film:

[0069] The treatment solution is a saturated potassium hydroxide anhydrous ethanol solution and acetone, with a mixing ratio of 2:1. The method of preparing the treatment solution is to add excess potassium hydroxide to anhydrous ethanol, seal with plastic wrap to prevent water from entering, heat in a 60°C water bath until the solution turns yellow, absorb 20ml of the supernatant into a test tube, and mix with acetone in a ratio of 2:1 to obtain a light yellow treatment solution. The treatment solution is stored in a 4°C refrigerator and can generally be stored for 2-3 days. Over time, the treatment solution will gradually turn brown and can continue to be used, but it will produce a pungent odor.

[0070] S5. Exhibition film:

[0071] Use a pipette to absorb 200ul of the treatment solution and add it to the slide. The treatment solution will spread rapidly. Use tweezers to pick up several slices and place them on the slide. Then immerse the slide completely in a 60℃ water tank. The slices immersed in water will float up rapidly and relax from being severely curled in the process.

[0072] S6, fish slices:

[0073] After the transferred slices float to the water surface, not all of them are intact. You need to pick the slices that look good to the naked eye, use a brush to gently move the slices so that part of the slices touch the new slide, and slowly lift the slide off the water surface. Generally, 1 / 3 to 1 / 2 of the transferred slices are good.

[0074] S7, baking sheet:

[0075] Transfer the slides to a 60°C oven for drying; the drying time must be longer than 2 hours. If the time is too short, the sections are prone to folding during staining. After being treated with the treatment solution, the sections will basically not overlap if the drying time is sufficient.

[0076] S8. Dyeing:

[0077] The preparation method of the staining solution is as follows:

[0078] ① Preparation of Coomassie Brilliant Blue G250 dye solution (dyeing protein in dark blue): 1 g Coomassie Brilliant Blue G250 + 400 ml ethanol + 70 ml glacial acetic acid to 1 L;

[0079] ② Preparation of eluent: 250 ml ethanol + 80 ml glacial acetic acid to 1 L;

[0080] ③ Coomassie Brilliant Blue G250 staining:

[0081] The steps for dyeing are as follows:

[0082] S81, placing the slices on a 60°C heating plate and heating until the slices are dry, the drying time is 30 minutes;

[0083] S82, add Coomassie Brilliant Blue G250 and stain for 2 min; specifically: use a pipette to draw 1 ml of Coomassie Brilliant Blue G250 onto the slide, shake it to evenly cover the slice, and stain for 2 min;

[0084] S83, eluent separation 10 min;

[0085] S84, wash with water for 1 min; dry the slices on a 60 ℃ heating plate; the drying time is 30 min;

[0086] S9, sealing: add a drop of sealing agent on the slice, then cover the cover slip and squeeze, the sealing agent will gradually spread to the whole slice. This step usually takes half an hour, and the standard for judging whether the sealing is completed is that the sealing agent basically spreads to the whole slice and the cover slip is firmly adhered.

[0087] Embodiment 1:

[0088] Taking the preparation of wheat resin embedded blocks and automated resin sectioning as an example, the specific method is as follows:

[0089] S1. Preparation of resin embedded blocks:

[0090] ① Pick 3 wheat grains with normal grain shape, soak them in water to soften them in advance; use a razor blade to cut a 2mm thick tissue block in the middle of the grain;

[0091] ② Use forceps to grab the tissue block and place it in a 2 ml centrifuge tube. Use a pipette to add 1.5 ml of fixative solution and immerse it in the fixative solution for 48 hours at room temperature to fully fix it.

[0092] ③ Use a pipette to remove the fixative solution, and use a pipette to draw 1.5 ml of 10× PBS buffer into the centrifuge tube to fully wash away the fixative solution. This step takes 20 minutes each time and is repeated 6 times;

[0093] ④ Use a step-by-step dehydration method to dehydrate the tissue blocks step by step according to the gradient of 25%, 50%, 75%, 100%, and 100%, with each step lasting 10 minutes; immediately add a new gradient ethanol solution after the original gradient ethanol solution is completely absorbed;

[0094] ⑤Permeate the tissue block step by step according to the gradient of 20%, 40%, 60%, 80%, 100%, and 100%; each level lasts 2 hours;

[0095] ⑥ Place the infiltrated tissue block with the cross section facing downward in a No. 1 transparent capsule, and use a pipette to slowly inject the resin until the tissue block is submerged. Prevent the generation of bubbles during the process. After the resin is injected, use tweezers to place the tissue block to prevent it from tilting;

[0096] ⑦ Place the embedding mold in a 60°C oven for 2 days to allow the resin to harden;

[0097] S2, repair block:

[0098] Use a microtome to trim the tissue block to a complete cross section.

[0099] S3, Slice:

[0100] ① Install the tungsten steel knife on the slicer and clamp it in the knife clamp;

[0101] ② Adjust the slice to the required thickness, and adjust the position of the blade and the embedding block so that the blade is aligned with the embedding block;

[0102] ③ Adjust the slicer to automatically slice, adjust the slicing speed, cut 30 slices, and pick up the slices for transfer;

[0103] ④ Place the slices in a 60 ℃ oven for 12 h to prevent them from falling off during the staining process;

[0104] S4. Pre-processing of the exhibition film:

[0105] Prepare the treatment solution by adding excess potassium hydroxide to anhydrous ethanol, seal with plastic wrap to prevent moisture from entering, heat in a 60°C water bath until the solution turns yellow, pipette 20 ml of the supernatant into a test tube, and mix with acetone in a ratio of 2:1.

[0106] S5. Exhibition film:

[0107] Use a pipette to absorb 200ul of the treatment solution and add it to the slide. The treatment solution will spread quickly. Use tweezers to pick up multiple slices and place them on the slide. Then immerse the slide completely in a 60℃ water tank. The slices immersed in water will float up quickly and unwind from severe curling in the process.

[0108] S5. Scoop out the slices:

[0109] After the transferred slices float to the water surface, pick out the slices that look better to the naked eye, use a brush to gently move the slices so that part of the slices come into contact with the slide, and slowly lift the slide off the water surface.

[0110] S7, baking sheet:

[0111] The slides were transferred to a 60°C oven for drying.

[0112] S8. Dyeing:

[0113] S81, place the slices on a 60 °C heating plate;

[0114] S82, add Coomassie Brilliant Blue G250 for staining for 2 min;

[0115] S83, eluent separation 10 min;

[0116] S84, wash with water for 1 min; dry on a heating plate and observe under a microscope;

[0117] S9. Sealing: Add a drop on the slice, then cover with a coverslip and squeeze. The sealing agent will gradually spread to the entire slice.

[0118] See Table 1 for a comparison of slicing speed and slicing quality between the slicing method of this embodiment and a known slicing method.

[0119] Table 1 Comparison of slicing speed and film quality of different slicing methods

[0120]

[0121] Based on Table 1, it can be seen that the slicing method of this embodiment is compared with the traditional resin slicing and the water tank slicing based on the working liquid recorded in the Chinese patent application, CN117517029B, and the slicing speed is qualitatively improved by 400%, and the slicing quality is high, and wrinkle-free and wrinkle-less slices can be prepared with high throughput. In addition, the slicing method of this embodiment can be adapted to machine automated slicing, which reduces the difficulty of getting started and saves labor resources.

[0122] Table 2 IamgeJ recognition results of slices prepared by different methods

[0123]

[0124] As can be seen from Table 2, the slices made by traditional manual resin slices are affected by wrinkles, the protein staining area increases, and the number and area of ​​wrinkles between different slices are inconsistent, the repeatability is poor, and the coefficient of variation is higher. The slices made by fully automatic slices without pressing greatly reduce the influence of wrinkles, the average protein staining area is lower, and the repeatability between different slices is good, and the coefficient of variation is lower.

[0125] See the instruction manual Figure 1-4 As shown in Table 1 and Table 2, the traditional resin sectioning method will produce wrinkles, which will affect the protein recognition effect, and the coefficient of variation shows that the number and area of ​​the wrinkles are different. The automatic sectioning of the present invention produces a stable staining area and eliminates the influence of wrinkles.

[0126] The parts not involved in the present invention are the same as the prior art or can be implemented by using the prior art.

[0127] As described above, although the present invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the present invention itself. Various changes in form and details may be made without departing from the spirit and scope of the present invention as defined in the appended claims.

Claims

1. An automated resin slicing method without tableting, characterized in that The following steps are involved: S1, preparing resin embedded blocks; S2, block trimming: Trim the resin embedded block prepared in S1, remove the excess resin around it, and trim a complete tissue block section; S3, slicing: without pressing, use a slicer to quickly cut a large number of slices of required thickness; collect severely curled slices; the slicing method is as follows: S31. Install a tungsten steel knife on the slicer and clamp it in the knife clamp; S32, adjusting the slice to a desired thickness, and adjusting the position of the blade and the resin embedding block so that the blade edge is aligned with the resin embedding block; S33, adjusting the slicer to automatically slice, adjusting the slicing speed, retracting the blade after slicing for a period of time, and clamping the adhered and severely curled slices, and clamping and transferring them together; S4, pretreatment of the slide: prepare a treatment solution according to the ratio of saturated potassium hydroxide anhydrous ethanol solution and acetone in 2:1, drop a small amount of the treatment solution onto the slide, and transfer the collected severely curled sections onto the slide; The preparation method of the treatment solution is to add excess potassium hydroxide to anhydrous ethanol, seal it with plastic wrap to prevent water from entering, heat it in a 60°C water bath until the solution turns yellow, draw 20 ml of the supernatant into a test tube, and mix it with acetone in a ratio of 2:1 to obtain a light yellow treatment solution; S5. Spread the slide: Insert the slide diagonally into the water tank quickly. The slice immersed in water will quickly float up and spread out. S6. Pick up the slices: Use a new slide to pick up the fully unfolded slices; S7, drying: transfer the glass slide in S6 to a 60°C oven for drying; S8, staining: stain the slices in S7 with a staining solution, and then transfer to a 60°C oven for drying after staining; S9, sealing: Use sealing medium to seal the slices in S8.

2. The method for automated resin slicing without tabletting according to claim 1, characterized in that: In S1, the preparation of resin embedding blocks is carried out according to experimental requirements, including but not limited to plant roots, stems or seeds; specifically, tissue blocks of a certain thickness are cut and the preparation of the embedding blocks is completed according to the resin section preparation process of fixation, dehydration and infiltration.

3. The method for automated resin slicing without tabletting according to claim 1, characterized in that: In S2, the block can be trimmed manually or automatically by a machine until the tissue block has a complete cross section.

4. The method for automated resin slicing without tabletting according to claim 1, characterized in that: In S33, the rotating arm on the slicer can be manually rotated to speed up the slicing process.

5. The method for automated resin slicing without tabletting according to claim 1, characterized in that: In S4, use a pipette to absorb 200ul of treatment solution and add it to the slide. The treatment solution will spread rapidly. Use tweezers to pick up multiple slices and place them on the slide so that each slice is in contact with the treatment solution. Then quickly immerse the slide completely in a 60℃ water tank. The slices immersed in water will float up rapidly and relax from being severely curled in the process.

6. The method for automated resin slicing without tabletting according to claim 1, characterized in that: After the slices transferred in S6 float to the water surface, not all of them are intact. You need to pick out the slices that look good to the naked eye, use a brush to gently move the slices so that part of the slices are in contact with the slide, and slowly lift the slide off the water surface.

7. The method for automated resin slicing without tabletting according to claim 1, characterized in that: The baking time in S7 and S8 was longer than 2 h.

8. The method for automated resin slicing without tabletting according to claim 1, characterized in that: In S9, a mounting medium is used. A drop is added to the slice, and then a coverslip is covered and squeezed. The mounting medium will gradually spread to the entire slice.

Images