Manufacturing method of paraffin section for research on development characteristics of rhizome and adventitious bud of bamboo reed
Through an improved paraffin sectioning method, including FAA fixation, 15% HF solution softening, ethanol dehydration, xylene clearing and paraffin embedding, the problem of unclear tissue in the microstructural study of Phragmites australis rhizomes and their adventitious buds was solved, the integrity and clarity of the sections were achieved, and the application of paraffin sectioning technology was promoted.
Patent Information
- Application Number
- CN202510687272.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-27
- Publication Date
- 2025-09-19
AI Technical Summary
Existing technologies make it difficult to effectively study the microstructural characteristics of Phragmites australis rhizomes and their adventitious buds. Paraffin sectioning technology is not effective when processing hard rhizomes and their adventitious buds, resulting in unclear tissue structure and difficulty in observing their development and differentiation process.
The samples were fixed with FAA fixative, followed by no dehydration, clearing, wax immersion and embedding, clearing, clearing, clearing, clearing, wax immersion and embedding, no dehydration, no dehydration, clearing, wax immersion and embedding, no dehydration, clearing, wax immersion and embedding. Combined with softening with 15% HF solution, dehydration with ethanol, clearing with xylene, paraffin embedding, and section staining, the section thickness and staining time were adjusted to ensure clear tissue structure.
The tissue structure integrity and clarity of paraffin sections of Phragmites australis rhizomes and their adventitious buds were achieved, which supported microstructural research and promoted the development and application of paraffin section technology.
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Figure CN120668432A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of paraffin sections, and in particular relates to a paraffin section preparation method for studying developmental characteristics of Phragmites australis rhizomes and adventitious buds thereof. Background Art
[0002] Arundo donax is a perennial rhizome plant belonging to the genus Arundo donax in the Poaceae family, widely distributed in tropical and temperate regions. High in protein, adaptable to a wide range of environments, and high in yield, Arundo donax is also known for its excellent resistance to biotic and abiotic stresses. It can be used as an energy plant and forage, as well as for saline-alkali land remediation and wastewater purification.
[0003] Phragmites australis is primarily propagated asexually, with its rhizomes serving as a crucial propagation material, significantly impacting population growth and biomass. However, current research on rhizomes and their adventitious buds is relatively limited, with even less understanding of their morphological and microstructural characteristics.
[0004] Paraffin sectioning is a common technique for studying morphological changes in plant cell tissues. However, the rigid rhizomes and adventitious buds of Phragmites australis are not easily accessible for wax immersion and sectioning. Therefore, strengthening the application of paraffin sectioning to the anatomical structure of Phragmites australis rhizomes and adventitious buds can provide technical support for studying the formation, development, and differentiation of rhizomes and adventitious buds. This can reveal the microscopic structure of Phragmites australis rhizomes and adventitious buds at a microscopic level, which is of great significance for the establishment of key anatomical databases and also facilitates the development and application of paraffin sectioning technology. Summary of the Invention
[0005] In view of this, the object of the present invention is to provide a paraffin section preparation method for studying the developmental characteristics of Phragmites australis rhizomes and their adventitious buds, which can ensure that the tissue structure of the obtained sections is clear and complete, and is convenient for observing the microstructure and developmental characteristics of Phragmites australis rhizomes and their adventitious buds.
[0006] To achieve the above object, the technical solution of the present invention is as follows:
[0007] A method for preparing paraffin sections for studying the developmental characteristics of Phragmites australis rhizomes and adventitious buds thereof comprises the following steps:
[0008] (1) Material collection: Select Phragmites australis rhizomes and their adventitious buds, clean them with water, and cut the samples into a size suitable for embedding boxes;
[0009] (2) Fixation: Fix the sample with FAA fixative for 48 h;
[0010] (3) Softening: Transfer the fixed sample to 15% HF solution and soften for 7 days;
[0011] (4) Dehydration: Dehydrate the sample step by step using different concentrations of ethanol;
[0012] (5) Transparency: The sample was treated with anhydrous ethanol-xylene mixed solution and xylene in sequence;
[0013] (6) Wax dipping and embedding: Transfer the transparent sample and xylene into a container in an oven, add paraffin wax until saturated to form a paraffin xylene saturated solution, and treat for 42-48 hours; then increase the oven temperature to 65°C, transfer the sample to the molten paraffin wax preheated to 65°C, treat for 4-8 hours, and finally embed;
[0014] (7) Sectioning, mounting, and drying: Cut the wax block into 10 μm strips, transfer the strips to 40°C water, allow them to spread on the water surface, then remove them with a clean glass slide and dry them in a 40°C drying machine for 2 h.
[0015] (8) Dewaxing: After the slides are dried, the paraffin on the sections is removed with xylene, and then the sections are rehydrated with ethanol of decreasing concentrations;
[0016] (9) Dyeing: Dyeing with safranin and fast green dyes;
[0017] (10) Sealing: After the slides were dried, they were sealed with neutral gum to obtain paraffin sections of Phragmites australis rhizomes and their adventitious buds.
[0018] Furthermore, in step (2), the FAA fixative comprises 70% ethanol solution, 40% formaldehyde solution and glacial acetic acid in a volume ratio of 90:5:5; and the volume of the FAA fixative is more than 20 times the volume of the Phragmites australis rhizome and its adventitious bud sample.
[0019] Furthermore, in step (4), dehydration is performed by treating with 70% ethanol for 2 h, and then sequentially treating with 85% ethanol, 95% ethanol, anhydrous ethanol, and anhydrous ethanol, each treatment lasting 1.5 h.
[0020] Furthermore, step (5) is transparent, and is sequentially treated with a mixture of anhydrous ethanol and xylene in a volume ratio of 1:1, xylene, and xylene, each treatment lasting 1 hour.
[0021] Furthermore, in step (6), when the paraffin wax is dissolved in xylene, the temperature of the oven is 37° C.; the operation of replacing the original paraffin wax in the wax cup with new paraffin wax preheated to a molten state is performed twice, and each treatment is performed for 2 hours.
[0022] Furthermore, the dewaxing in step (8) is carried out by sequentially treating with xylene, xylene, a mixture of anhydrous ethanol and xylene in a volume ratio of 1:1, anhydrous ethanol, anhydrous ethanol, 95% ethanol, 85% ethanol, and 70% ethanol, each treatment lasting 5 minutes.
[0023] Furthermore, in step (9), after staining with safranin dye for 70 minutes, the sample was rinsed with distilled water for 10 seconds; then treated with 70% ethanol, 85% ethanol, 95% ethanol, and 95% ethanol in sequence, each for 1 minute; dyed with fast green dye for 10 seconds; then treated with 95% ethanol, anhydrous ethanol, and anhydrous ethanol in sequence, each for 1 minute; finally, treated in a mixture of anhydrous ethanol and xylene with a volume ratio of 1:1, xylene, and xylene in sequence, each for 5 minutes.
[0024] The beneficial effects of the present invention include at least:
[0025] The present invention uses 15% HF solution to soften rhizomes and adventitious buds, thereby preventing sample tissue cells from collapsing and deforming; appropriately increases the thickness of slices to effectively avoid wrinkles or damage to the slices; and adjusts the staining time to ensure uniform staining and clear tissue structure.
[0026] The present invention adjusts a conventional paraffin section preparation method to solve the shortcomings and deficiencies in the technology, ensures that the tissue cells of the prepared Arundo donax paraffin sections are complete and clear, is conducive to observing the microstructure of Arundo donax rhizomes and adventitious buds, can provide reliable technical support for process research on the formation, development and differentiation of Arundo donax rhizomes and adventitious buds, is conducive to the comprehensive utilization of Arundo donax, and is also conducive to the development and application of paraffin section technology. BRIEF DESCRIPTION OF THE DRAWINGS
[0027] Figure 1 Schematic diagram of the microstructure of Phragmites australis rhizome, where A is a panoramic view and B is a partial view.
[0028] Figure 2 Schematic diagram of the microstructure of adventitious buds on the rhizome of Phragmites australis, where A is a panoramic view and B is a partial view.
[0029] Figure 3 Schematic diagram of paraffin sections obtained by softening treatment in the present invention, wherein A is a panoramic view and B is a partial view.
[0030] Figure 4 Schematic diagram of paraffin sections obtained by section thickness in the present invention, wherein A is a panoramic view and B is a local view.
[0031] Figure 5 This is a schematic diagram of paraffin sections obtained using a staining time for a comparative example, wherein A is a panoramic view and B is a local view.
[0032] Figure 6 This is another schematic diagram of paraffin sections obtained using a staining time obtained in Comparative Example 3, wherein A is a panoramic view and B is a local view. Specific implementation methods
[0033] The following is a detailed description of the solution proposed by the present invention with reference to the accompanying drawings:
[0034] (1) Material collection: Select Phragmites australis rhizomes, clean them with water, and cut them into pieces suitable for embedding boxes;
[0035] (2) Fixation: Fix the sample with FAA fixative (70% ethanol solution: 40% formaldehyde solution: glacial acetic acid = 90:5:5) for 48 h; the volume of FAA fixative should be at least 20 times the volume of the sample;
[0036] (3) Softening: The fixed samples were transferred to 15% HF solution for softening for 7 days;
[0037] (4) Dehydration: Treat with 70% ethanol for 2 h, then sequentially treat with 85% ethanol, 95% ethanol, anhydrous ethanol, and anhydrous ethanol, each treatment for 1.5 h;
[0038] (5) Transparency: The sample was treated with a mixture of anhydrous ethanol and xylene at a volume ratio of 1:1, xylene, and xylene, respectively, for 1 h each treatment.
[0039] (6) Wax dipping and embedding: Transfer the transparent material and xylene into a container in an oven, add paraffin wax until saturated to form a paraffin xylene saturated solution. The oven temperature is 37°C and the treatment is carried out for 42-48 hours. Then, the oven temperature is raised to 65°C, and the sample is transferred to the molten paraffin wax preheated to 65°C for 4 hours, replacing it once in the middle. Finally, embedding;
[0040] (7) Sectioning, mounting, and drying: Cut the wax block into 10 μm strips, transfer the strips to 40°C water, allow them to spread on the water surface, then remove them with a clean glass slide and dry them in a 40°C drying machine for 2 h.
[0041] (8) Dewaxing: After the slides are dried, they are treated with xylene twice, each for 5 min; then treated with a mixture of anhydrous ethanol and xylene (volume ratio 1:1), anhydrous ethanol, anhydrous ethanol, 95% ethanol, 85% ethanol, and 70% ethanol, each for 5 min;
[0042] (9) Staining: After staining with safranin for 70 minutes, rinse with distilled water for 10 seconds; then treat with 70% ethanol, 85% ethanol, 95% ethanol, and 95% ethanol in sequence, each for 1 minute; after staining with fast green for 10 seconds, treat with 95% ethanol, anhydrous ethanol, and anhydrous ethanol in sequence, each for 1 minute; finally, treat with a mixture of anhydrous ethanol and xylene with a volume ratio of 1:1, xylene, and xylene in sequence, each for 5 minutes;
[0043] (10) Mounting: After the slides are dried, they are mounted with neutral gum to obtain paraffin sections of Phragmites australis rhizomes;
[0044] (11) Observe and take photos under a biological microscope, such as Figure 1 As shown, the tissue structure is intact and the staining is clear.
[0045] Example 2
[0046] (1) Material collection: Select adventitious buds of Phragmites australis rhizomes, clean them with water, and cut them into pieces suitable for embedding boxes;
[0047] (2) Fixation: Fix the sample with FAA fixative (70% ethanol solution: 40% formaldehyde solution: glacial acetic acid = 90:5:5) for 48 h; the volume of FAA fixative should be at least 20 times the volume of the sample;
[0048] (3) Softening: The fixed samples were transferred to 15% HF solution for softening for 7 days;
[0049] (4) Dehydration: Treat with 70% ethanol for 2 h, then sequentially treat with 85% ethanol, 95% ethanol, anhydrous ethanol, and anhydrous ethanol, each treatment for 1.5 h;
[0050] (5) Transparency: The sample was treated with a mixture of anhydrous ethanol and xylene at a volume ratio of 1:1, xylene, and xylene, respectively, for 1 h each treatment.
[0051] (6) Wax dipping and embedding: Transfer the transparent material and xylene into a container in an oven, add paraffin wax until saturated to form a paraffin xylene saturated solution. The oven temperature is 37°C and the treatment is carried out for 42-48 hours. Then, the oven temperature is raised to 65°C, and the sample is transferred to the molten paraffin wax preheated to 65°C for 4 hours, replacing it once in the middle. Finally, embedding;
[0052] (7) Sectioning, mounting, and drying: Cut the wax block into 10 μm strips, transfer the strips to 40°C water, allow them to spread on the water surface, then remove them with a clean glass slide and dry them in a 40°C drying machine for 2 h.
[0053] (8) Dewaxing: After the slides are dried, they are treated with xylene twice, each for 5 min; then treated with a mixture of anhydrous ethanol and xylene (volume ratio 1:1), anhydrous ethanol, anhydrous ethanol, 95% ethanol, 85% ethanol, and 70% ethanol, each for 5 min;
[0054] (9) Staining: After staining with safranin for 70 minutes, rinse with distilled water for 10 seconds; then treat with 70% ethanol, 85% ethanol, 95% ethanol, and 95% ethanol in sequence, each for 1 minute; after staining with fast green for 10 seconds, treat with 95% ethanol, anhydrous ethanol, and anhydrous ethanol in sequence, each for 1 minute; finally, treat with a mixture of anhydrous ethanol and xylene with a volume ratio of 1:1, xylene, and xylene in sequence, each for 5 minutes;
[0055] (10) Mounting: After the slides are dried, they are mounted with neutral gum to obtain paraffin sections of adventitious buds of Phragmites australis rhizomes;
[0056] (11) Observe and take photos under a biological microscope, such as Figure 2 As shown, the tissue structure is intact and the staining is clear.
[0057] Since the rhizomes and adventitious buds of Phragmites australis at different developmental stages can be prepared for paraffin sectioning using this method, and the paraffin section preparation effects of rhizomes and adventitious buds of rhizomes in different comparative examples are the same, only the paraffin section preparation of rhizomes of Phragmites australis will be used as a comparative example in the following, and the comparative example of the paraffin section preparation of adventitious buds of Phragmites australis rhizomes will no longer be presented.
[0058] About softening treatment:
[0059] Paraffin sections were prepared after softening treatments of no softening treatment, 15% HF treatment for 3 days, 15% HF treatment for 5 days, 15% HF treatment for 7 days, 15% HF treatment for 15 days, 15% HF treatment for 30 days, 15% HF treatment for 90 days, 50% alcohol:50% glycerol treatment for 7 days, and 20% HCl treatment for 1 day. Other steps were the same as those in Example 1. The effects of different softening treatments on the preparation of paraffin sections of Phragmites australis rhizomes were compared. The results are shown in Table 1.
[0060] Table 1 Effects of different softening treatment methods on the preparation of paraffin sections of Phragmites australis rhizomes
[0061]
[0062]
[0063] As shown in Table 1, when the tissue was not softened or softened with 50% alcohol: 50% glycerol, 20% HCl, the preparation effect was poor, the tissue collapsed and broke, and it was impossible to slice. When softened with 15% HF for 3 days, the preparation results were as follows. Figure 3 As shown, the processing time is not enough, the softening is not enough, the tissue structure is damaged after sectioning, and the structure inside the epidermis is empty; when 15% HF is used to soften for 5 days, the preparation results are the same as Figure 1Similarly, the tissue structure was damaged after sectioning, and the structure inside the epidermis was empty; when softened with 15% HF for 7 days, the preparation results were as follows Figure 1 As shown, the tissue structure is complete and clear; when softened with 15% HF for 15 days, the preparation results are the same as Figure 1 Similar, the tissue structure is complete and clear; when softened with 15% HF for 30 days, the preparation results are the same as Figure 1 When softened with 15% HF for 90 days, the sample became brittle and could not be used for the next experiment. After comprehensive comparison, the present invention selected 15% HF softening for 7 days to prepare paraffin sections of Phragmites australis rhizomes.
[0064] Different slice thicknesses:
[0065] The thickness of the paraffin sections was set to 5 μm, 10 μm, and 15 μm for paraffin sectioning. Other steps were the same as in Example 1. The effects of different section thicknesses on the preparation of paraffin sections of Phragmites australis rhizomes were compared. The results are shown in Table 2.
[0066] Table 2 Effects of different slice thicknesses on the preparation of paraffin sections of Phragmites australis rhizomes
[0067] serial number thickness Slice effect 1 5μm The tissue is shrunken and folded, and cannot be unfolded 2 10μm Complete and clear organizational structure 3 15μm Complete and clear organizational structure
[0068] As shown in Table 2, when the slice thickness is set to 5 μm, the preparation results are as follows Figure 4 As shown, the tissue is wrinkled and folded, and it is difficult to unfold the slice, making it difficult to observe the complete slice structure; when the slice thickness is set to 10μm, the preparation results are as follows Figure 1 As shown, the tissue structure is complete and clear; when the slice thickness is set to 15μm, the preparation results are the same as Figure 1 The tissue structure is complete and clear. For comprehensive comparison, the present invention sets the slice thickness to 10 μm for paraffin section preparation of Phragmites australis rhizome.
[0069] Dyeing time:
[0070] Safranin Fast Green is a commonly used reagent for xylem and phloem staining, and different staining times have a significant impact on the staining effect. Therefore, this comparative example subjected paraffin sections to three treatments: safranin staining for 70 minutes and Fast Green staining for 10 seconds, safranin staining for 70 minutes and Fast Green staining for 30 seconds, and safranin staining for 150 minutes and Fast Green staining for 10 seconds. Other method steps were the same as in Example 1. The effects of different staining times on the preparation effect of Arundo donax paraffin sections were compared, and the results are shown in Table 3.
[0071] Table 3 Effects of different staining times on the preparation of paraffin sections of Phragmites australis rhizomes
[0072] serial number Safranin staining time Fast green dyeing time Dyeing effect 1 70 minutes 10s Clear and distinct colors 2 70 minutes 30s Uneven dyeing and too dark fast green 3 150min 10s Uneven staining and too dark safranin staining
[0073] As shown in Table 1, the results of staining with safranin dye for 70 minutes and fast green dye for 10 seconds are as follows: Figure 1 As shown, the staining effect is good and the tissue cell structure is clear; when stained with safranin dye for 70 minutes and fast green dye for 30 seconds, the preparation results are as follows Figure 5 As shown, the staining is uneven, the vascular bundle and parenchyma tissue show too dark fast green staining, the cell boundaries are not clear, and the tissue cell structure cannot be distinguished; when stained with safranin dye for 150 minutes and fast green dye for 30 seconds, the preparation results are as follows Figure 6 As shown, the staining is uneven, the vascular bundle and parenchyma tissue show that the safranin staining is too dark, the cell boundaries are unclear, and the tissue cell structure cannot be distinguished. Comprehensive comparison, the present invention selects to use safranin staining for 70 minutes and fast green staining for 10 seconds to prepare paraffin sections of Phragmites australis rhizomes.
[0074] In summary, the present invention improves the preparation steps of paraffin sections of Phragmites australis rhizomes and their adventitious buds, including softening with a 15% HF solution for 7 days, selecting a paraffin section thickness of 10 μm, staining with safranin for 70 minutes, and staining with Fast Green for 10 seconds. The present invention fully preserves the cellular arrangement and tissue structure characteristics of the Phragmites australis rhizomes and their adventitious buds, and can produce sections with complete and clear tissue structure and distinct staining, thus providing an excellent method for preparing paraffin sections of Phragmites australis.
[0075] The above describes the preferred embodiments of the present invention. It should be understood that the present invention is not limited to the specific embodiments described above. A person skilled in the art may make various improvements and modifications without departing from the principles of the present invention, and such improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. A method for preparing paraffin sections for studying the developmental characteristics of Phragmites australis rhizomes and their adventitious buds, characterized in that: The following steps are involved: (1) Material collection: Select Phragmites australis rhizomes and their adventitious buds, clean them with water, and cut the samples into a size suitable for embedding boxes; (2) Fixation: Fix the sample with FAA fixative for 48 h; (3) Softening: Transfer the fixed sample to 15% HF solution and soften for 7 days; (4) Dehydration: Dehydrate the sample step by step using different concentrations of ethanol; (5) Transparency: The sample was treated with anhydrous ethanol-xylene mixed solution and xylene in sequence; (6) Wax dipping and embedding: Transfer the transparent sample and xylene into a container in an oven, add paraffin wax until saturated to form a paraffin xylene saturated solution, and treat for 42-48 hours; then increase the oven temperature to 65°C, transfer the sample to the molten paraffin wax preheated to 65°C, treat for 4-8 hours, and finally embed; (7) Sectioning, mounting, and drying: Cut the wax block into 10 μm strips, transfer the strips to 40°C water, allow them to spread on the water surface, then remove them with a clean glass slide and dry them in a 40°C drying machine for 2 h. (8) Dewaxing: After the slides are dried, the paraffin on the sections is removed with xylene, and then the sections are rehydrated with ethanol of decreasing concentrations; (9) Dyeing: Dyeing with safranin and fast green dyes; (10) Sealing: After the slides are dried, they are sealed with neutral gum to obtain paraffin sections of Phragmites australis rhizomes and their adventitious buds.
2. The paraffin section preparation method for studying the developmental characteristics of Phragmites australis rhizomes and adventitious buds according to claim 1, characterized in that: In step (2), the FAA fixative comprises 70% ethanol solution, 40% formaldehyde solution and glacial acetic acid in a volume ratio of 90:5:5; and the volume of the FAA fixative is more than 20 times the volume of the Phragmites australis rhizome sample.
3. The paraffin section preparation method for studying the developmental characteristics of Phragmites australis rhizomes and adventitious buds according to claim 2, characterized in that: Step (4) Dehydration: Treat with 70% ethanol for 2 h, then sequentially treat with 85% ethanol, 95% ethanol, anhydrous ethanol, and anhydrous ethanol, each treatment for 1.5 h.
4. The method for preparing paraffin sections for studying the developmental characteristics of Phragmites australis rhizomes and adventitious buds according to claim 3, characterized in that: Step (5) is transparent, which is treated in sequence with a mixture of anhydrous ethanol and xylene in a volume ratio of 1:1, xylene, and xylene, each treatment lasting 1 hour.
5. The method for preparing paraffin sections for studying the developmental characteristics of Phragmites australis rhizomes and their adventitious buds according to claim 4, characterized in that: In step (6), when paraffin is dissolved in xylene, the oven temperature is 37° C.; the operation of transferring the sample into molten paraffin preheated to 65° C. is performed twice, with each treatment lasting 2 h.
6. The method for preparing paraffin sections for studying the developmental characteristics of Phragmites australis rhizomes and their adventitious buds according to claim 5, characterized in that: The dewaxing step (8) is carried out by sequentially treating with xylene, xylene, a mixture of anhydrous ethanol and xylene in a volume ratio of 1:1, anhydrous ethanol, anhydrous ethanol, 95% ethanol, 85% ethanol, and 70% ethanol, with each treatment lasting 5 minutes.
7. The method for preparing paraffin sections for studying the developmental characteristics of Phragmites australis rhizomes and adventitious buds according to any one of claims 1 to 6, characterized in that: In step (9), after staining with safranin dye for 70 minutes, rinse with distilled water for 10 seconds; then treat with 70% ethanol, 85% ethanol, 95% ethanol, and 95% ethanol in sequence, each for 1 minute; stain with fast green dye for 10 seconds; then treat with 95% ethanol, anhydrous ethanol, and anhydrous ethanol in sequence, each for 1 minute; finally, treat in a mixture of anhydrous ethanol and xylene with a volume ratio of 1:1, xylene, and xylene in sequence, each for 5 minutes.