A method for preparing a paraffin section of a woody dicotyledon root system

Abstract

The present application belongs to the technical field of paraffin section, and particularly relates to a preparation method of a woody dicotyledon root system paraffin section. The preparation method of the woody dicotyledon root system paraffin section provided by the present application comprises the following steps in sequence: fixing, sampling, dehydration and pre-staining, secondary transparency, wax immersion, embedding, block trimming, cementing, sectioning, section spreading and sticking, dewaxing, rehydration, staining, dehydration and transparency. The preparation method of the present application is closely matched with each step, and is connected with the previous step and the next step. The preparation method of the present application can significantly improve the success rate of root system tissue sectioning while shortening the sectioning time, and the section is complete, clear and easy to observe, which can effectively solve the technical problem that the plant root system of different development degrees and the regional structure difference are too large to be sectioned in the prior art.

Description

Technical Field

[0001] This invention belongs to the field of paraffin sectioning technology, specifically relating to a method for preparing paraffin sections of the roots of woody dicotyledonous plants. Background Technology

[0002] Roots are vital organs for plant absorption of water and nutrients, as well as for nutrient synthesis, and they possess the ability to respond to both abiotic and biotic signals. The structure of plant roots is closely related to their physiological functions. Changes in root structure can induce alterations in a series of physiological and biochemical processes, including mycorrhizal symbiosis, root morphology, root mechanics, root respiration and secretion, nitrogen fixation symbiosis, and resource absorption, ultimately impacting plant growth to a certain extent. Therefore, the structure of plant root cell tissues is of great significance for studying plant adaptive mechanisms and can provide a theoretical basis and practical guidance for plant cultivation and management.

[0003] Paraffin sectioning has long been a common technique for studying morphological changes in plant cells and tissues. However, the structural differences between different developmental stages of plant roots are significant. Young tissues have higher water content and are more fragile, making their cell structure easily deformed during slide preparation. Mature tissues have relatively well-developed xylem and a denser structure, making them prone to insufficient wax impregnation and slide failure, both of which hinder the observation of the microstructure of root tissues. Based on these problems, this invention improves upon traditional paraffin sectioning techniques, establishing a method for preparing paraffin sections of woody dicotyledonous plant roots with intact cell structures. This method effectively solves the problem of difficulty in preparing slides due to significant structural differences between different developmental stages of plant roots, and is of great significance for the study of plant root cell structure. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing paraffin sections of roots from woody dicotyledonous plants. This method comprehensively improves upon conventional paraffin sectioning techniques, including dehydration, clearing, wax impregnation, sectioning, and embedding. The technical solution of this invention is easier to operate, saves experimental time, significantly improves the success rate of root tissue preparation, and produces sections with complete and clear cellular structures, facilitating in-depth research on the cellular structure of plant roots at different developmental stages.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A method for preparing paraffin sections of the roots of woody dicotyledonous plants includes the following steps in sequence: fixation, sampling, dehydration and pre-staining, secondary clearing, paraffin impregnation, embedding, trimming, bonding, sectioning, spreading and bonding, dewaxing, rehydration, staining, dehydration, and clearing.

[0007] The fixation and sampling process involves fixing the roots of woody dicotyledonous plants in a formaldehyde-glacial acetic acid-ethanol solution, followed by sampling.

[0008] The dehydration and pre-staining process involved sequentially treating the root samples with 70% ethanol, 85% ethanol + safranin, 90% ethanol, 95% ethanol, 100% ethanol, and 100% ethanol to complete the dehydration and pre-staining treatments. The 85% ethanol + safranin treatment lasted for 2 hours, and the concentration of safranin in 85% ethanol was 1 g / 100 mL.

[0009] The secondary transparency process involves pre-treating the dehydrated and pre-stained root samples by soaking them in a mixture of anhydrous ethanol and xylene, followed by soaking them in xylene to complete the secondary transparency treatment. The soaking time in the mixture is 1 hour, and the xylene soaking is performed twice, with each soaking lasting 1 hour.

[0010] The wax impregnation process involves first impregnating the root samples after they have undergone secondary transparency in a mixed wax solution, and then replacing the mixed wax solution with a beeswax-paraffin mixture for further impregnation. The mixed wax solution is composed of xylene and paraffin wax fragments.

[0011] The embedding process involves first adding a portion of the beeswax-paraffin mixture from the wax impregnation process to the embedding box. Before the beeswax-paraffin mixture on the surface of the embedding box solidifies, the remaining beeswax-paraffin mixture, including the root sample, is added to complete the embedding process. The embedding box has a length, width, and height of 3.8-4.2 cm, 2.6-3 cm, and 0.3-0.7 cm, respectively, and is made of sulfuric acid paper.

[0012] The adhesion process involves attaching the trimmed root sample to the slide base; the slide base has a hollow cylindrical three-dimensional structure with an inner diameter of 1.8-2.2 cm at the bottom and a height of 0.5-1.5 cm.

[0013] The slicing process involves slicing the cemented root samples, controlling the slice thickness to match the degree of root development. Specifically, when the root sample is from the root apical tissue region, the slice thickness is 11-13 μm; when the root sample is from a highly developed non-root apical tissue region, the slice thickness is 15-17 μm.

[0014] The dewaxing process involves immersing the root samples after spreading and gluing them in xylene for 25 minutes, and then replacing the xylene with fresh xylene and immersing for another 10 minutes.

[0015] The rehydration process involves treating the dewaxed root samples with ethanol at different concentration gradients in stages; the ethanol gradients are 100%, 90%, 80%, 70%, and 50% respectively.

[0016] Furthermore, the sampling length is a root segment of 0.8-1.2 cm.

[0017] Furthermore, in the dehydration and pre-staining process, the treatment time for 70% ethanol, 90% ethanol, 95% ethanol, 100% ethanol, and 100% ethanol is 40 minutes.

[0018] Furthermore, the specific process of the wax impregnation is as follows: First, the transparent root sample is placed in a mixture of xylene and paraffin wax (volume ratio 1:1) at 63-65℃ for 12 hours; Second, the mixed wax solution is replaced with a beeswax-paraffin mixture, and the impregnation continues for 60 hours; After the mixed wax solution is replaced with a beeswax-paraffin mixture for impregnation, it is replaced 5 times with a beeswax-paraffin mixture; The beeswax content in the beeswax-paraffin mixture is 8 wt%.

[0019] Furthermore, in the embedding process, the amount of beeswax-paraffin mixture added first is enough to completely cover the bottom of the embedding box.

[0020] Furthermore, in the rehydration process, ethanol of different concentration gradients is used for step-by-step rehydration treatment, with each step taking 3 minutes.

[0021] Furthermore, the spreading and gluing process involves placing the wax strip of the root sample obtained after slicing onto a glass slide, and then placing the glass slide on a spreading stage to complete the spreading and gluing process; the spreading and gluing time is 50-70 minutes.

[0022] Furthermore, the staining involves placing the rehydrated root sample in a 1% toluidine blue aqueous solution for staining; the staining time is 3 minutes.

[0023] Further, the dehydration involves treating the stained root samples sequentially with 50% ethanol, 70% ethanol, 80% ethanol, 90% ethanol, 100% ethanol, and 100% ethanol, with each treatment lasting 3 minutes; the clearing involves immersing the dehydrated root samples in xylene for 1 minute.

[0024] Furthermore, the prepared slides do not require mounting and can be directly observed under a microscope.

[0025] Compared with the prior art, the beneficial effects of this invention are as follows:

[0026] This invention provides a method for preparing paraffin sections of the roots of woody dicotyledonous plants, which significantly improves the success rate of section preparation, mainly in the following aspects:

[0027] (1) The wax impregnation time has been increased, which can ensure that the root samples with a high degree of lignification have sufficient wax impregnation time and effectively prevent the stele tissue from detaching from the wax strip when the root samples with a high degree of lignification are sliced.

[0028] (2) The wax impregnation uses a mixture of beeswax and paraffin wax. Beeswax can effectively reduce the hardness of the wax block, which is beneficial to obtaining a relatively flat sample during the block trimming and slicing process.

[0029] (3) After the sample is impregnated with wax, it is added to the embedding box in two separate times. This can not only avoid the wax block layering phenomenon, but also effectively enhance the hardness of the root cell tissue, which is conducive to subsequent sectioning operations.

[0030] (4) Adding pre-staining can make the sample in the embedding stage visible in the opaque wax block, making it easier to observe the sample position and effectively avoid cell damage during the block trimming process; in addition, the pre-staining dye is safranin, which can still make the epidermis, endodermis and xylem cells appear light red after staining with toluidine blue dye, which is helpful to distinguish the cell tissue structure of the root cross section.

[0031] This invention provides a method for preparing paraffin sections of the roots of woody dicotyledonous plants, which saves experimental time, mainly in the following aspects:

[0032] The secondary transparenting time is 3 hours, which saves approximately 1.5 to 3 hours compared to the conventional transparenting time. The secondary transparenting time set by this invention ensures thorough transparenting of root samples at different developmental stages, while also preventing root samples with lower developmental stages from becoming hardened and brittle due to prolonged transparenting time.

[0033] Furthermore, the slide base used in this invention has a hollow cylindrical three-dimensional structure. This structure ensures that the positions of multiple samples remain consistent, reducing the frequency of microtome adjustments and minimizing machine wear. Moreover, this invention controls the slide thickness to match the root development level of the root sample, effectively preventing damage to root cross-sectional tissue and cell walls caused by excessively thin or thick slides, which can lead to unclear cell boundaries. Furthermore, the embedding cassette is made of tracing paper, which does not easily adhere to paraffin, facilitating the removal of paraffin blocks. The tracing paper is also reusable, reducing experimental costs. Finally, rehydration is performed using multiple ethanol gradients stepwise, avoiding cell deformation caused by large differences in ethanol concentration, which could lead to slide preparation failure.

[0034] In summary, this invention comprehensively improves upon existing techniques in terms of dehydration, clearing, paraffin impregnation, embedding, sectioning, and dewaxing. Each step is closely coordinated and seamlessly integrated, effectively addressing the technical challenge of preparing tissue sections from plant roots at different developmental stages and with significant regional structural differences. Furthermore, the preparation method of this invention is simple to operate, saves experimental time, and significantly improves the success rate of root tissue section preparation. The resulting sections are complete, clear, and easy to observe, facilitating research on the structure of plant roots at different developmental stages. Attached Figure Description

[0035] Figure 1This is a paraffin section micrograph of the root system of *Sapium sebiferum* 60 days after growth, located 0.5 cm from the root tip.

[0036] Figure 2 This is a paraffin section of the root system of *Sapium sebiferum* 1 cm from the root tip, taken after 60 days of growth in Example 2.

[0037] Figure 3 This is a paraffin section micrograph of the root system of *Sapium sebiferum* 60 days after growth, located 1.5 cm from the root tip.

[0038] Figure 4 This is a paraffin section micrograph of the root system of *Sapium sebiferum* 2 cm from the root tip after 60 days of growth, as shown in Example 4.

[0039] Figure 5 This is a paraffin section micrograph of the root system at the middle position of the primary lateral root of *Sapium sebiferum* after 60 days of growth, as shown in Example 5.

[0040] Figure 6 This is a paraffin section micrograph of a primary lateral root system of *Sapium sebiferum* 60 days after growth, located 2 cm from the main root.

[0041] Figure 7 This is a comparison diagram of root samples from Example 1 (without pre-staining) and Example 1 (with pre-staining);

[0042] Figure 8 The images are microscopic images of paraffin-embedded root sections with controlled section thicknesses of 5 and 8 μm, respectively, in Comparative Example 2.

[0043] Figure 9 The images are microscopic images of paraffin-embedded root sections with controlled section thicknesses of 22 and 20 μm, respectively, in Comparative Example 3.

[0044] Figure 10 The image shown is a microscopic image of a paraffin section of root tip prepared using the conventional clarification method in Comparative Example 4.

[0045] Figure 11 This is a microscopic image of a paraffin section of a primary lateral root at the middle position, prepared using the conventional clarification method in Example 5.

[0046] Figure 12 This is a microscopic image of a paraffin section of a root system in Comparative Example 6, in which the beeswax-paraffin mixture was replaced with paraffin.

[0047] Figure 13 Comparative Example 7 shows microscopic images of paraffin-embedded root sections at different developmental stages obtained by adjusting the paraffin immersion time to 36 h and 48 h, respectively.

[0048] Figure 14 This image shows a situation where air bubbles are prone to occur during slide preparation using conventional embedding methods.

[0049] Figure 15These are comparison images of the slice bases: a wooden block and a centrifuge tube cap. Detailed Implementation

[0050] The technical solution of the present invention will be further explained below with reference to specific embodiments and accompanying drawings.

[0051] Unless otherwise specified, the raw materials and preparation methods used in the following embodiments are conventional materials and techniques in the art.

[0052] The slicer used was a Leica RM2235.

[0053] Based on the laws of cell growth and development, this invention sets the sampling gradient to different developmental stages of cell tissue along the vertical axis, thereby obtaining paraffin sections of root cells at different developmental stages.

[0054] Example 1

[0055] This embodiment uses the woody dicotyledonous plant *Sapium sebiferum* as an example to illustrate the technical solution and effects of the present invention, but these should not be construed as limiting the scope of protection of the present invention. Specifically, the material selected in this embodiment is root tissue of *Sapium sebiferum* grown for 60 days in Longting District, Kaifeng City. The preparation method of paraffin sections includes the following steps:

[0056] Sampling: The roots of the Chinese tallow tree preserved in formaldehyde-glacial acetic acid-ethanol solution (FAA fixative) were removed with tweezers. The root tips of the primary lateral roots were placed upwards, and the cell tissue of the root segment 0.5 cm away from the root tip was taken with a blade. After sampling, the sample was placed in a 70% ethanol solution for later use. The FAA fixative consisted of 70% ethanol, glacial acetic acid and formaldehyde in a volume ratio of 18:1:1.

[0057] Dehydration and pre-staining: Root tip samples were sequentially immersed in 70% ethanol for 40 min, then in 85% ethanol + safranin solution for 2 h, and then sequentially immersed in 90%, 95%, 100%, and 100% ethanol solutions for 40 min each to complete dehydration and pre-staining; the concentration of safranin in 85% ethanol was 1 g / 100 mL.

[0058] Secondary transparency: The dehydrated and pre-stained root tip samples were immersed in a mixture of anhydrous ethanol and 100% xylene at a volume ratio of 1:1 for 1 hour, and then immersed twice in 100% xylene for 1 hour each time.

[0059] Wax impregnation: Pour out half of the xylene from the container, add wax shavings with a melting point of 60-62℃ to fill the container to form a mixed wax solution. Place the transparent root tip sample and the mixture of wax solution in an oven at 63℃ for 12 hours. Then replace the mixed wax solution with a beeswax-paraffin mixture to fill the container. Replace the beeswax-paraffin mixture every 3 hours during the day (at 12:00, 15:00, and 18:00 respectively), for a total of 6 replacements. The wax impregnation time is 60 hours.

[0060] Embedding: Use tracing paper to fold a rectangular embedding box with a length, width, and height of 4cm, 2.8cm, and 0.5cm respectively. Place the embedding box on a 60℃ slide stage. First, add some beeswax-paraffin mixture to completely cover the bottom of the embedding box. When the wax at the bottom turns white (the surface beeswax-paraffin mixture has not solidified), add the remaining wax liquid, including the root tip sample. Use tweezers to adjust the position of the sample so that the root tip sample is neatly embedded in the paraffin. After it has completely solidified, peel off the wax block.

[0061] Trimming and bonding: Cut out root tip samples of appropriate size according to the position of the sample (leave a blank circle to make it easier to adjust the position, size and shape of the sample); then attach the trimmed root tip samples to the centrifuge tube cap to complete the bonding process.

[0062] Sectioning, spreading and mounting: Sections were prepared using a microtome, with the section thickness controlled at 12 μm. Then, an appropriate amount of adhesive was applied to the slide using a toothpick and spread evenly with lens paper. After that, 1 mL of deionized water was added. The wax strip of the cut root tip sample was placed on the slide using tweezers and then placed on a 42℃ spreading stage for 1 hour.

[0063] Dewaxing and rehydration: The root tip samples after spreading and gluing were sequentially immersed in 100% xylene for 25 min, and then immersed in fresh 100% xylene for 10 min to complete the dewaxing treatment; The dewaxed root tip samples were sequentially immersed in anhydrous ethanol and xylene solution (1:1 volume ratio), 100% ethanol, 90% ethanol, 80% ethanol, 70% ethanol, and 50% ethanol for 3 min each to complete the rehydration treatment;

[0064] Staining: Immerse the rehydrated root tip sample in a 1% toluidine blue aqueous solution for 3 min;

[0065] Dehydration: Use deionized water to gently rinse away excess stain around the root tip sample after staining. Then, immerse the stained root tip sample in ethanol with volume concentrations of 50%, 70%, 80%, 90%, 100%, and 100% for 3 minutes each to complete the dehydration process.

[0066] Transparency: The dehydrated root tip sample was immersed in 100% xylene solution for 1 minute to obtain paraffin sections of root tip cell tissue.

[0067] Example 2

[0068] The difference between Example 2 and Example 1 is that the sampling site is different. A 1cm root segment cell tissue is taken from 1cm away from the root tip using a blade. The rest of the steps are the same as in Example 1 to prepare root paraffin sections.

[0069] Example 3

[0070] The difference between Example 3 and Example 1 is that the sampling site is different. A 1cm root segment cell tissue is taken from 1.5cm away from the root tip using a blade. The rest of the steps are the same as in Example 1 to prepare root paraffin sections.

[0071] Example 4

[0072] The difference between Example 4 and Example 1 is that the sampling site is different. A 1cm root segment cell tissue is taken from 2cm away from the root tip using a blade. The rest of the steps are the same as in Example 1 to prepare root paraffin sections.

[0073] Example 5

[0074] The difference between Example 5 and Example 1 is that the sampling site is different. The sampling site is the middle position of the primary lateral root, and the slice thickness is 16μm. The rest of the steps are the same as in Example 1 to obtain root paraffin slices.

[0075] Example 6

[0076] The difference between Example 6 and Example 1 is that the sampling site is different. The sampling site is a 1cm root segment cell tissue of the primary lateral root 2cm away from the main root. The section thickness is 16μm. The rest of the steps are the same as in Example 1 to prepare root paraffin sections.

[0077] Comparative Example 1

[0078] The difference between Comparative Example 1 and Example 1 is that no pre-staining treatment was performed, but the rest of the steps were the same as in Example 1, and root paraffin sections were obtained.

[0079] Comparative Example 2

[0080] The difference between Comparative Example 2 and Example 1 is that the slice thickness is different. The slice thickness was controlled to be 5 μm and 8 μm respectively, and the rest of the steps were the same as in Example 1 to obtain root paraffin slices.

[0081] Comparative Example 3

[0082] The difference between Comparative Example 3 and Example 6 is the thickness of the sections. The section thicknesses were controlled to be 22 and 20 μm, respectively, while the rest of the steps were the same as in Example 6, to obtain root paraffin sections.

[0083] Comparative Example 4

[0084] The difference between Comparative Example 4 and Example 1 is that the conventional transparent method is used. The specific transparent process is as follows: the dehydrated and pre-stained root samples are first soaked in a mixture of anhydrous ethanol and xylene for 1.5 hours, and then soaked twice in 100% xylene for 1.5 hours each time. The rest of the steps are the same as in Example 1, and root paraffin sections are obtained.

[0085] Comparative Example 5

[0086] The difference between Comparative Example 5 and Example 6 is that the conventional transparent method is used. The specific transparent process is as follows: the dehydrated and pre-stained root samples are first soaked in a mixture of anhydrous ethanol and xylene for 1.5 hours, and then soaked twice in 100% xylene for 1.5 hours each time. The rest of the steps are the same as those in Example 6, and root paraffin sections are obtained.

[0087] Comparative Example 6

[0088] The difference between Comparative Example 6 and Example 1 is that: during the wax impregnation process, the beeswax-paraffin mixture was replaced with paraffin to fill the container, while the rest of the steps were the same as in Example 1, and root paraffin slices were obtained.

[0089] Comparative Example 7

[0090] The difference between Comparative Example 7 and Example 6 is that the root samples after secondary transparency were directly immersed in a beeswax-paraffin mixture for 36 hours and 48 hours respectively. The rest of the steps were the same as those in Example 6, and root paraffin sections were obtained.

[0091] Microscopic examination results

[0092] Paraffin sections prepared in Examples 1-6 and Comparative Examples 1-7 were observed and images were acquired using a Leica upright microscope.

[0093] Figure 1-6 The images show paraffin sections of root cell tissues at different developmental stages prepared in Examples 1-6. As can be seen from the figures, the paraffin sections prepared from the undifferentiated root samples of Example 1, the differentiated root samples of Example 2, and the root samples at secondary xylem development stages I-IV of Examples 3-6 all exhibit uniform staining, intact cell structure, and clear cell boundaries. This indicates that the preparation method of the present invention is applicable to root samples at different developmental stages.

[0094] Figure 7 To compare the results of Example 1 (without and with pre-staining treatment), images of root tip samples during the embedding process and microscopic images of paraffin sections of the prepared root tip cell tissue are provided. Figure 7The image on the left (A) shows the root sample without pre-staining, while the image on the right (B) shows the root sample with pre-staining. Comparing the two images, it can be seen that the position and direction of the roots are clearly visible in the pre-stained root sample, making it easier to observe. This can avoid cell damage during the trimming process and improve the success rate of slide preparation. Figure 7 B Figure 7 C represents microscopic images of paraffin sections stained with toluidine blue only (without pre-staining) and those stained with toluidine blue after pre-staining, respectively. Observation Figure 7 According to BC, pre-staining can make the root epidermis, endodermis, and xylem vessels appear light red, which is helpful in distinguishing the cellular tissue structure of the root cross section.

[0095] Figure 8 Comparative Example 2 shows microscopic images of root paraffin sections with different section thicknesses. Figure 8 Images A and B are, in order: microscopic images of paraffin sections of secondary lateral root tips with a section thickness of 5 μm and microscopic images of paraffin sections of secondary lateral root tips with a section thickness of 8 μm. Figure 9 Microscopic images of root paraffin sections with controlled section thicknesses of 22 and 20 μm for Comparative Example 3. Figure 9 Images A and B are, respectively, microscopic images of paraffin sections from the primary lateral root maturity zone with a section thickness of 22 μm and 20 μm, respectively. Figure 8-9 It is known that sections that are too thin or too thick can damage the root cross-section tissue, and cell wall damage can make the boundaries between cells unclear. Moreover, sections that are too thick can cause cell overlap, resulting in ghosting and making it impossible to observe a clear cross-section.

[0096] Figure 10 For Comparative Example 4, a paraffin section of the root tip was prepared using a conventional clarification method and examined under a microscope. Figure 11 For Comparative Example 5, a conventional clearing method was used to obtain a paraffin section microscopic image of the root system located midway between the base and tip of the primary lateral root. Figure 10-11 It is known that increasing the transparency time has little impact on roots with a high degree of development, but for root tip samples with a low degree of development, the root sample tissue is prone to hardening and becoming brittle due to excessively long transparency time, making it difficult to cut complete sections.

[0097] Figure 12 The image shows a microscopic examination of a paraffin section obtained by replacing the beeswax-paraffin mixture with paraffin in Comparative Example 6. As can be seen from the image, the cellular structure of the root sample is incomplete. This is because sections prepared using only paraffin often exhibit incomplete cellular structures due to the hardness of the wax block. In contrast, the beeswax-paraffin mixture used in this invention softens the wax block after impregnation, making the sectioning process smoother and significantly reducing the frequency of incomplete cellular structures in the resulting sections, thus improving the success rate of slide preparation.

[0098] Figure 13 To modify the paraffin impregnation process in Comparative Example 7, root samples that had undergone secondary clearing were directly immersed in a beeswax-paraffin mixture. Paraffin sections were prepared for microscopic examination after immersion for 36 hours and 48 hours, respectively. Figure 13 A is a wax impregnation process that lasts 36 hours. Figure 13 B shows a paraffin micrograph of a root sample that has been paraffin-impregnated for 48 hours. As can be seen from the figure, insufficient paraffin impregnation time causes the stele tissue of highly lignified root samples to detach from the paraffin strip during sectioning, leading to sectioning failure. Therefore, a paraffin impregnation time shorter than that specified in this invention is suitable for young roots but not for highly lignified root samples.

[0099] Figure 14 The images show the results of a conventional embedding method, where a beeswax-paraffin mixture containing the sample is directly poured into the embedding cassette, with the sample position adjusted using tweezers or a dissecting needle. As the images show, this embedding method frequently generates air bubbles, preventing further steps and requiring the experiment to be restarted. Therefore, it significantly increases the experimental time.

[0100] Figure 15 The images show a comparison of using wooden blocks and centrifuge tube caps as slice bases. As can be seen, using wooden blocks as slice bases is difficult because it's hard to ensure uniform block size, thus requiring multiple adjustments to the microtome. In this embodiment, however, centrifuge tube caps are used as slice bases. This is advantageous because the sample length is 1cm, which matches the height of the cap, effectively ensuring a consistent sample height after loading. This avoids multiple adjustments to the microtome, saving manpower, reducing machine wear, and minimizing the risk to operators from repeated adjustments. Furthermore, centrifuge tube caps are readily available in the laboratory.

[0101] In summary, through a comprehensive comparison of various factors, it is evident that the improved paraffin sectioning method of this invention saves preparation time and significantly improves the success rate. This method is suitable for preparing paraffin sections of the roots of woody dicotyledonous plants. It is easier to operate and produces complete and clear cellular tissue sections, which is of great significance for the study of plant root structure.

[0102] The above are merely preferred embodiments of the present invention and are not limited to the examples described above. Those skilled in the art will recognize that various modifications and variations can be made based on the principles of the present invention. Any modifications or improvements made should be considered within the scope of protection of the present invention.

Claims

1. A method for preparing paraffin sections of woody dicotyledonous plant root systems, characterized in that, Comprise the following steps in sequence: Fixing, sampling, dehydration and pre-staining, secondary transparency, wax immersion, embedding, block repairing, fixing, slicing, section spreading and sticking, dewaxing, rehydration, staining, dehydration, transparency; The fixing and sampling are that the woody dicotyledon root system is placed in a formaldehyde-glacial acetic acid-ethanol solution for fixing, and after completion, sampling is performed; The dehydration and pre-staining are that the sampled root system sample is sequentially subjected to 70% ethanol treatment, 85% ethanol+safranin treatment, 90% ethanol treatment, 95% ethanol treatment, 100% ethanol treatment, and 100% ethanol treatment to complete the dehydration and pre-staining treatment; the 85% ethanol+safranin treatment time is 2 h; the safranin concentration in 85% ethanol is 1 g / 100 mL; In the dehydration and pre-staining, the 70% ethanol treatment, 90% ethanol treatment, 95% ethanol treatment, 100% ethanol treatment, and 100% ethanol treatment time is 40 min; The secondary transparency is that the root system sample after dehydration and pre-staining is first immersed in a mixture of anhydrous ethanol and xylene for pretreatment, and then immersed in xylene to complete the secondary transparency treatment; the mixture immersion time is 1 h, and the xylene immersion is performed twice, with each time being 1 h; The wax immersion is that the root system sample after secondary transparency is first subjected to wax immersion treatment in a mixed wax solution, and then the mixed wax solution is replaced with a beeswax-paraffin wax mixed solution to continue the wax immersion treatment; the mixed wax solution is composed of xylene and paraffin wax chips; The specific process of the wax immersion is that, in the first step, the transparent root system sample is placed in a mixture of xylene and paraffin wax chips with a volume ratio of 1:1 to form a mixture, and the mixture is treated at 63-65°C for 12 h; in the second step, the mixed wax solution is replaced with a beeswax-paraffin wax mixed solution, and the wax immersion is continued for 60 h; after the wax immersion treatment using the mixed wax solution, the mixed wax solution is replaced 5 times using a beeswax-paraffin wax mixed solution; in the beeswax-paraffin wax mixed solution, the beeswax content is 8 wt%; The embedding is that, in the embedding box, part of the beeswax-paraffin wax mixed solution in the wax immersion process is first added, and when the beeswax-paraffin wax mixed solution on the surface of the embedding box has not solidified, the remaining beeswax-paraffin wax mixed solution containing the root system sample is added to complete the embedding treatment; the length, width, and height of the embedding box are 3.8-4.2 cm, 2.6-3 cm, and 0.3-0.7 cm, respectively, and the material is sulfuric acid paper; The fixing is that the block-repaired root system sample is adhered to the section base; the section base has a hollow cylindrical three-dimensional structure, and the inner diameter of the bottom surface of the hollow cylinder is 1.8-2.2 cm, and the height is 0.5-1.5 cm; The slicing is that the fixed root system sample is sliced, and the slice thickness is controlled to match the root system development degree of the root system sample; when the root system sample is a root tip tissue area, the slice thickness is 11-13 μm; when the root system sample is a non-root tip tissue area with high development degree, the slice thickness is 15-17 μm; The dewaxing is that the section spreading and sticking root system sample is immersed in xylene for 25 min, and then new xylene is replaced for continued immersion for 10 min; The rehydration is carried out by using different concentration gradients of ethanol to rehydrate the dewaxed root system sample; the ethanol gradients are 100%, 90%, 80%, 70% and 50% in sequence.

2. A method for preparing paraffin sections of woody dicotyledonous plant root systems according to claim 1, characterized in that, The length of the sample is 0.8-1.2 cm.

3. A method for preparing paraffin sections of woody dicotyledonous plant root systems according to claim 1, characterized in that, In the embedding, the amount of the first added mixture of beeswax and paraffin is used to completely cover the bottom of the embedding box.

4. A method for preparing paraffin sections of woody dicotyledonous plant root systems according to claim 1, characterized in that, In the rehydration, different concentration gradients of ethanol are used to rehydrate the root system sample, and the time for each rehydration is 3 min.

5. A method for preparing paraffin sections of woody dicotyledonous plant root systems according to claim 1, characterized in that, The sectioning and sticking are carried out by placing the wax ribbon of the obtained root system sample after sectioning on a glass slide and placing the glass slide on a sectioning and sticking table; the time for the sectioning and sticking is 50-70 min.

6. A method for preparing paraffin sections of woody dicotyledonous plant root systems according to claim 1, characterized in that, The staining is carried out by placing the rehydrated root system sample in 1% aqueous toluidine blue; the staining time is 3 min.

7. A method for preparing paraffin sections of woody dicotyledonous plant root systems according to claim 1, characterized in that, The dehydration is carried out by sequentially treating the stained root system sample with 50% ethanol, 70% ethanol, 80% ethanol, 90% ethanol, 100% ethanol and 100% ethanol, and the time for each treatment is 3 min; the transparency is achieved by immersing the dehydrated root system sample in xylene; the time for the xylene immersion is 1 min.

8. A method for preparing paraffin sections of woody dicotyledonous plant root systems according to any one of claims 1 to 7, characterized in that, The prepared section does not need to be sealed and can be directly observed under a microscope.

Images