Histological staining method for phyllostachys plants and method for distinguishing phloem cells

Abstract

The invention provides a histological staining method of phyllostachys plants and a method for distinguishing phloem cells, and belongs to the technical field of tissue slices. The invention provides a histological staining method of a phyllostachys plant, which is characterized in that histological staining is carried out by using an antibody generated by trimethylated histone, a tissue slice is taken as a sample, and the antibody is expressed in other cells except the phloem and is subjected to chromogenic reaction, but is not expressed in the phloem and is free from any coloration. Therefore, the method disclosed by the invention can be used for specifically staining different tissue cells, is high in sensitivity and low in cost, can be used for judging whether the cells are phloem cells or not by utilizing a common electron microscope according to whether cell walls and / or cell nucleuses are colored or not, and the cell nucleuses are clearly visible due to the fact that the cell walls of the phloem are not colored. The histological staining method disclosed by the invention is particularly suitable for detecting morphological characteristics of cell nucleuses of early-stage phloem cells.

Description

Technical Field

[0001] The present invention belongs to the technical field of tissue sectioning, and particularly relates to a histological staining method for plants of the genus Phyllostachys and a method for differentiating phloem cells. Background Art

[0002] Bamboo plants belong to the genus Phyllostachys of the Gramineae family. The vascular tissue is scattered in the ground tissue. The vascular tissue of plants is composed of various tubular molecules that make up the xylem and phloem, including xylem initial cells, protoxylem vessels, tracheids, sieve tubes, companion cells, etc. For bamboo shoots in the initial and middle stages of development, various tubular cells are in a state of differentiation, and the specialized structures of the phloem such as sieve plates have not yet formed or are in the process of formation. In addition, at this time, the number of differentiated phloem cells is small, and they are difficult to identify because they are similar in shape, size, and structure to adjacent parenchyma cells. Importantly, the process of phloem cell differentiation is accompanied by a process of apoptosis, and the morphology of the cell nucleus changes significantly, which is an important indicator for characterizing the development process of sieve elements.

[0003] Currently, for bamboo and most plants, qualitative histological staining methods are used to distinguish by comparing the differences in the colors of cell walls. For example, after safranin-fast green staining, the cell walls of sieve elements in the phloem are dark blue, and the thickened parts of the cell walls in the xylem are brick red. However, during conventional staining, since the main components of the cell walls of various tissues are basically the same, all types of cell walls are stained, only the degree of staining is different, resulting in problems of poor specificity and low sensitivity. At the same time, due to the coloring of the cell walls, it is difficult to observe the size, morphology, and distribution of cell nuclei in phloem cells.

[0004] In addition, although the use of a transmission electron microscope (abbreviated as TEM) can observe the ultrastructure of cells, including cell organelles, cytoplasm, and cell nuclei, providing more information for differentiating tissue development and cell types; however, compared with samples embedded in conventional paraffin, TEM samples take a long time, have cumbersome steps, and are difficult to operate; the reagent cost is high; moreover, the cost of TEM equipment is high and it has not been popularized yet. Summary of the Invention

[0005] The present invention provides a histological staining method for plants of the genus Phyllostachys and a method for differentiating phloem cells, which can specifically stain tissues, and phloem cells can be differentiated by color under an ordinary electron microscope, and the internal structural characteristics of the phloem can be clearly observed.

[0006] The present invention provides a histological staining method for plants of the genus Phyllostachys, comprising the following steps: preparing a tissue section from the bamboo shoot tissue in the development period, using the tissue section as a sample, incubating successively with an antibody against trimethylated histone, a secondary antibody, and a DAB chromogenic solution, then counterstaining with hematoxylin, dehydrating with alcohol, clearing with xylene, and mounting for microscopic observation.

[0007] In a preferred embodiment of the present invention, the bamboo shoot bud tissue includes removing the bamboo shoot buds in the development stage from the nodes of the underground stem, and cutting the middle part or the bamboo shoot sheaths of the bamboo shoot bodies into pieces.

[0008] In a preferred embodiment of the present invention, the bamboo shoot bud tissue is successively subjected to tissue fixation, dehydration, clearing, infiltration with paraffin wax, embedding, sectioning, baking of the sections, and dewaxing of the sections to water to prepare tissue sections.

[0009] In a preferred embodiment of the present invention, before incubation with the antibody against trimethylated histone, antigen repair, inactivation of endogenous enzymes, and serum blocking are also included.

[0010] In a preferred embodiment of the present invention, the antigen repair includes subjecting the tissue sections to heat-induced antigen repair in an EDTA antigen repair solution, and the composition of the EDTA antigen repair solution is Tris-EDTA; The inactivation of endogenous enzymes includes adding a 3% (v / v) H2O2 solution to the tissue sections; The serum blocking includes dropping 5% (v / v) goat serum onto the tissue sections and then allowing them to stand at room temperature.

[0011] In a preferred embodiment of the present invention, the antibody against trimethylated histone includes an antibody generated from trimethylated histone H3.

[0012] In a preferred embodiment of the present invention, the antibody against trimethylated histone includes anti-H3K4me3.

[0013] In a preferred embodiment of the present invention, the dilution ratio of the anti-H3K4me3 is 1:50 to 200.

[0014] The present invention also provides the application of the above histological staining method in differentiating the phloem cells of plants of the genus Phyllostachys.

[0015] The present invention also provides a method for differentiating the phloem cells of plants of the genus Phyllostachys, which includes staining tissue sections using the above histological staining method and then observing under a microscope, where the early phloem cells are not stained and the remaining cells are stained.

[0016] Beneficial effects: The present invention provides a histological staining method for plants of the genus Phyllostachys, which uses an antibody generated by trimethylated histone for histological staining. Taking tissue sections as samples, the antibody is expressed in other cells except phloem, and color reactions occur, and the staining degree depends on the amount of the antibody expressed in the tissue. In the cell wall and / or nucleus, it shows brown, yellowish-brown or sepia. However, the antibody is not expressed in the phloem and there is no coloring. Therefore, the method of the present invention can specifically stain different tissue cells, has high sensitivity and low cost. By using an ordinary electron microscope, it can be determined whether it is a phloem cell according to whether the cell wall and / or nucleus is colored. Moreover, since the cell wall of the phloem is not colored, the nucleus is clearly visible. The histological staining method of the present invention is particularly suitable for detecting the morphological characteristics of the nucleus of early phloem cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Microscopic examination image of the staining result of Example 1 (scale bar: 50 μm); Figure 2 Microscopic examination image of the staining result of the control group of Example 1 (scale bar: 50 μm); Figure 3 Microscopic examination image of the staining result of Example 2 (scale bar: 50 μm); Figure 4 Microscopic examination image of the staining result of the control group of Example 2 (scale bar: 50 μm); Figure 5 Microscopic examination image of the staining result of Example 3 (scale bar: 50 μm); Figure 6 Microscopic examination image of the staining result of the control group of Example 3 (scale bar: 50 μm); Figure 7 Microscopic examination image of the staining result of Comparative Example 1 (scale bar: 50 μm); Figure 8 Microscopic examination image of the staining result of Comparative Example 2 (scale bar: 100 μm). DETAILED DESCRIPTION OF THE INVENTION

[0018] The present invention provides a histological staining method for plants of the genus Phyllostachys, which includes the following steps: preparing tissue sections from shoot bud tissues in the developmental stage, taking the tissue sections as samples, incubating them successively with an antibody of trimethylated histone, a secondary antibody and a DAB chromogenic solution, and then performing counterstaining with hematoxylin, dehydrating with alcohol and clearing with xylene before mounting and observing under a microscope.

[0019] The present invention uses tissue sections as samples, and there are no special limitations on the preparation method of the tissue sections. For example, the selected tissue is successively subjected to tissue fixation, dehydration, clearing, infiltration with paraffin wax, embedding, sectioning, baking, and dewaxing of the sections to water to prepare tissue sections. When preparing the tissue sections, the methods in the literature can be referred to, such as the methods of You Peilei (You Peilei, Xu Jie, Qian Zhongying. Generation and section observation of callus of Abelmoschus esculentus. Acta Agriculturae Shanghai, 2007, (03): 92-95) or Zhang Qiaoying (Zhang Qiaoying, Yao Genyou. Preparation method and skills of paraffin tissue microarray. The Practical Journal of Cancer, 2005 (06), 551-553).

[0020] In the embodiments of the present invention, histological staining was carried out taking bamboo as an example. Among them, the tissue sections were prepared using bamboo shoot bud tissue, and the bamboo shoot bud tissue included taking the bamboo shoot buds in the development period from the nodes of the underground stem, cutting the middle part of the bamboo shoot body into pieces, or cutting the bamboo shoot sheath into pieces. The development period in the present invention includes the early development stage, the middle development stage, and the late development stage. In the upper and middle parts of the bamboo shoot buds in the early development stage, the cells constituting the vascular tissue are elongated to varying degrees; at this time, the procambium has differentiated into protoxylem and is strongly lignified, while the specialization of phloem is not obvious; in the upper and middle parts of the bamboo shoot buds in the middle development stage, the end walls of sieve elements are significantly specialized; in the upper and middle parts of the bamboo shoot buds in the late development stage, both sieve elements and companion cells in the phloem are significantly specialized. Compared with the vascular tissue in the internode part adjacent to the node where it is located, the vascular tissue cells in the culm sheath part differentiate earlier.

[0021] After the corresponding tissue sections are prepared in the present invention, antigen repair, inactivation of endogenous enzymes, and serum blocking are required before the incubation of the primary antibody and the secondary antibody can be carried out.

[0022] For antigen repair of the tissue sections in the present invention, in specific operations, the tissue sections are immersed in an EDTA antigen repair solution and heated in a microwave oven at medium fire (60 °C) for 8 min, the fire is stopped for 5 min, and then at low fire (30 °C) for 5 min, and then cooled to room temperature. The composition of the EDTA antigen repair solution in the present invention includes Tris-EDTA. When preparing, 10 mL of 20×Tris-EDTA (pH 8.0) is mixed with 190 mL of distilled water to obtain a 1× antigen repair solution with a pH of 8.0 containing 1 mM EDTA. After cooling to room temperature in the present invention, it is also necessary to wash with 0.01 M PBS (pH 7.2-7.6), 5 min each time, and wash 2 times in total.

[0023] After the above washing, the present invention performs an operation to inactivate endogenous enzymes, such as adding an H2O2 solution to the above tissue section and allowing it to stand at room temperature for a period of time. In one embodiment, an H2O2 solution with a volume percentage of 3% is added and allowed to stand at room temperature for 10 min; then it is washed again with the above PBS, 5 min each time, for a total of 2 washes.

[0024] The present invention seals the tissue section after enzyme inactivation. Serum can be used for sealing, such as 5% goat serum in the embodiment. During the specific operation, 5% goat serum is dropped onto the section and placed in a wet box at room temperature for 30 min; then it is washed with 0.01M PBS (pH 7.2 - 7.6), 5 min each time, for a total of 2 washes.

[0025] The present invention drops a primary antibody onto the sealed section for incubation. The primary antibody is a polyclonal antibody generated from trimethylated histone. In the embodiment, the antibody generated after trimethylation of histone H3, such as anti-H3K4me3, is a rabbit polyclonal antibody purchased from Abbkine Biotechnology Co., Ltd., and its product number is: A22225. When dropping the primary antibody, the primary antibody needs to be diluted, and the dilution ratio is 1:50 - 200, such as it can be 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:110, 1:120, 1:130, 1:140, 1:150, 1:160, 1:170, 1:180, 1:190 or 1:200. After dropping the appropriately diluted primary antibody, incubation is required. The incubation temperature is 4°C and the time is 8 - 12 h. In the embodiment, after dropping the primary antibody, it is placed in a 4°C refrigerator overnight.

[0026] The present invention takes out the slide after incubation with the primary antibody, rewarms it in a 37°C incubator for 30 min; rinses it with PBS, 5 min each time, for a total of 2 rinses; then performs secondary antibody incubation. 50 - 100 μL of the secondary antibody is dropped onto the slide and incubated in a 37°C incubator for 30 min. After the incubation, it is rinsed again with PBS, 5 min each time, for a total of 2 rinses. The secondary antibody used in the embodiment of the present invention is HRP-labeled goat anti-rabbit IgG (Servicebio, GB23303, dilution ratio is 1:200).

[0027] The present invention performs DAB color development on the slide after secondary antibody incubation. 50 - 100 μL of the pre-prepared DAB working solution (0.05%, w / v) is dropped onto the slide and incubated at room temperature for 1 - 15 min, and the reaction time is controlled according to the color development.

[0028] After the DAB color development, the present invention needs to perform counterstaining, that is, first wash the slide after DAB color development with water, and then soak it in 0.5% w / v hematoxylin for counterstaining for 1 - 3 min, and wash with water to turn blue.

[0029] After the counterstaining in the present invention, dehydration is carried out. The dehydration is performed step by step using alcohols with gradient concentrations, and the volume percentage content of the alcohol solution used increases successively to achieve the purpose of dehydration. The volume percentage content of the alcohol solution used is 60 - 100%, and the concentration of each stage can be 60%, 70%, 80%, 90% and 100%, and dehydration is carried out for 5 minutes at each stage.

[0030] The present invention performs a clearing operation on the dehydrated glass slide, such as placing it in xylene for 10 minutes, and then sealing the slide with neutral balsam, and then it can be observed and photographed under an electron microscope.

[0031] The present invention also provides an application of the above histological staining method in differentiating phloem cells of plants of the genus Phyllostachys.

[0032] When the specialized structures such as sieve plates of phloem cells such as sieve elements have not yet formed or are in the process of formation, based on the fact that phloem cells are not stained by the antibody, the present invention can distinguish the unstained early phloem cells (including sieve mother cells, developing sieve elements and companion cells) from the stained other tissues, including xylem, epidermis and ground tissue composed of parenchyma cells, etc.; at the same time, since the phloem is not stained, the cell morphology of phloem cells including sieve elements and companion cells, the size, number and morphology of the cell nucleus, and its distribution position in the cell, etc., can all be clearly observed with the aid of a conventional electron microscope.

[0033] The present invention also provides a method for differentiating phloem cells of plants of the genus Phyllostachys, which includes staining tissue sections using the above histological staining method and then observing under a microscope. The early phloem cells are not stained, and the remaining cells are stained.

[0034] When differentiating using the method of the present invention, the cell walls and cell nuclei of the tubular elements of the phloem, including sieve elements and companion cells, are not stained at all, while the cell walls and / or cell nuclei of other cells except the phloem are colored brown, yellow - brown or brownish - brown.

[0035] In order to further illustrate the present invention, the following examples are used to describe in detail a histological staining method for plants of the genus Phyllostachys and a method for differentiating phloem cells provided by the present invention, but they should not be construed as limiting the protection scope of the present invention.

[0036] Example 1 1. Shoot bud collection: Use pruning shears commonly used in horticulture to remove the shoot buds in the early stage of development from the nodes of the underground stem, cut the shoot body into small pieces of 0.5 cm × 0.5 cm, and select the small pieces located in the upper part of the shoot body for tissue fixation.

[0037] 2. Tissue fixation, dehydration, clearing, infiltration with paraffin wax, embedding, sectioning, baking the sections, dewaxing the sections to water.

[0038] 3. Antigen heat repair: Immerse the sections in the EDTA antigen repair solution, heat in a microwave oven at medium power for 8 min, turn off the fire for 5 min, then heat at low power for 5 min, and cool to room temperature. After cooling, wash with 0.01M PBS (pH 7.2 - 7.6) for 5 min × 2 times.

[0039] 4. Add 3% H2O2 and incubate at room temperature for 10 min to inactivate endogenous enzymes. Rinse with PBS for 5 min × 2 times.

[0040] 5. Serum blocking: Drop 5% goat serum on the sections and place them in a humid box at room temperature for 30 min. Wash with PBS for 5 min × 2 times; 6. Incubate with the primary antibody: Drop the appropriately diluted primary antibody anti - H3K4me3 (A22225, rabbit polyclonal antibody, Abbkine Scientific Co., Ltd., Wuhan, China), (dilution ratio 1:100), and drop an equal amount of PBS in the control group, and incubate overnight in a 4℃ refrigerator.

[0041] 7. Incubate with the secondary antibody: Take the slides out of the refrigerator and warm them at 37℃ in an incubator for 30 min; Rinse with PBS for 5 min × 2 times; Drop 100 μL of the secondary antibody and incubate at 37℃ in an incubator for 30 min; Rinse with PBS for 5 min × 2 times.

[0042] 8. DAB color development: Take the slides out of the incubator, rinse with PBS for 5 min × 2 times; Drop 50 - 100 μL of the pre - prepared 0.05% DAB working solution for color development and incubate at room temperature for 15 min.

[0043] 9. Counterstaining: Wash the slides after color development, then soak them in 0.5% hematoxylin for counterstaining for 3 min, and wash with water to turn blue.

[0044] 10. Dehydration: Dehydrate with alcohols of different concentrations (60%, 70%, 80%, 90%, 100%) for 5 min for each concentration.

[0045] 11. Clearing: Place the samples in xylene for 10 min after taking them out.

[0046] 12. Sealing the slides: Seal the slides with neutral balsam and observe under a microscope.

[0047] 13. Photograph the results.

[0048] The results of the experimental group are as Figure 1 shown. There is no staining in the cell walls and nuclei of the sieve - tube elements in the phloem of the upper part of the bamboo shoot body, which is consistent with the negative control. The colors of the cell walls and / or nuclei of other cells except the phloem are brown, yellow - brown or brownish - black. The results of the control group are as Figure 2As shown, the cell nuclei of all cell types are blue, and the cell walls are not significantly stained.

[0049] Example 2 1. Bamboo shoot bud collection: Use pruning shears commonly used in horticulture to remove the bamboo shoot buds in the mid-development stage from the nodes of the underground stem. Cut the bamboo shoot sheaths into small pieces of 0.5 cm × 0.5 cm, and select the small pieces located in the upper part of the bamboo shoot body for tissue fixation.

[0050] 2. Tissue fixation, dehydration, clearing, infiltration with paraffin wax, embedding, sectioning, baking the sections, and dewaxing the sections to water.

[0051] 3. Heat antigen repair: Immerse the sections in the EDTA antigen repair solution, heat them in a microwave oven at medium power for 8 min, stop the heating for 5 min, then heat at low power for 5 min, and cool to room temperature. After cooling, wash with 0.01M PBS (pH 7.2 - 7.6) for 5 min × 2 times.

[0052] 4. Add 3% H2O2 and incubate at room temperature for 10 min to inactivate endogenous enzymes. Rinse with PBS for 5 min × 2 times.

[0053] 5. Serum blocking: Drop 5% sheep serum on the sections and place them in a humid box at room temperature for 30 min. Wash with PBS for 5 min × 2 times; 6. Incubate with the primary antibody: Drop the appropriately diluted primary antibody anti-H3K4me3 (A22225, rabbit polyclonal antibody, Abbkine Scientific Co., Ltd., Wuhan, China), (dilution ratio 1:100), and drop an equal amount of PBS in the control group, and incubate overnight in a 4°C refrigerator.

[0054] 7. Incubate with the secondary antibody: Take the slides out of the refrigerator and warm them in an incubator at 37°C for 30 min; rinse with PBS for 5 min × 2 times; drop 100 μL of the secondary antibody and incubate in an incubator at 37°C for 30 min; rinse with PBS for 5 min × 2 times.

[0055] 8. DAB color development: Take the slides out of the incubator, rinse with PBS for 5 min × 2 times; drop 100 μL of the pre-prepared 0.05% DAB working solution for color development and incubate at room temperature for 1 - 15 min.

[0056] 9. Counterstaining: Wash the slides after color development, then immerse them in 0.5% hematoxylin for counterstaining for 3 min, and wash with water to turn blue.

[0057] 10. Dehydration: Dehydrate with alcohols of different concentrations (60%, 70%, 80%, 90%, 100%) for 5 min for each concentration.

[0058] 11. Clearing: Place the samples in xylene for 10 min after taking them out.

[0059] 12. Sealing the slides: Seal the slides with neutral balsam and observe under a microscope.

[0060] 13. Take pictures of the results.

[0061] The results of the experimental group are as Figure 3 shown. The cell walls and nuclei of the sieve elements and companion cells in the phloem of the bamboo shoot shell part showed no staining, which was consistent with the negative control. The cell walls and / or nuclei of other cells except the phloem were brown, yellowish-brown or brownish-black. The results of the control group are as Figure 4 shown. The nuclei of all types of cells were blue, and the cell walls showed no obvious staining.

[0062] Example 3 1. Collection of bamboo shoot buds: Use pruning shears commonly used in horticulture to remove the bamboo shoot buds in the late development stage from the nodes of the underground stem, cut the bamboo shoot body into small pieces of 0.5 cm × 0.5 cm, and select the small pieces located in the upper part of the bamboo shoot body for tissue fixation.

[0063] 2. Tissue fixation, dehydration, clearing, infiltration with paraffin wax, embedding, sectioning, baking the sections, and dewaxing the sections to water.

[0064] 3. Heat-induced antigen retrieval: Immerse the sections in the EDTA antigen retrieval solution, heat them in a microwave oven at medium power for 8 min, stop heating for 5 min, then heat at low power for 5 min, and cool to room temperature. After cooling, wash with 0.01 M PBS (pH 7.2 - 7.6) for 5 min × 2 times.

[0065] 4. Add 3% H2O2 and incubate at room temperature for 10 min to inactivate endogenous enzymes. Rinse with PBS for 5 min × 2 times.

[0066] 5. Serum blocking: Drop 5% goat serum on the sections and place them in a humid box at room temperature for 30 min. Wash with PBS for 5 min × 2 times; 6. Incubate with the primary antibody: Drop the appropriately diluted primary antibody anti-H3K4me3 (A22225, rabbit polyclonal antibody, Abbkine Biotechnology Co., Ltd., Wuhan, China), (dilution ratio 1:100), and drop an equal amount of PBS in the control group, and incubate overnight in a 4°C refrigerator.

[0067] 7. Incubate with the secondary antibody: Take the slides out of the refrigerator and warm them at 37°C in an incubator for 30 min; rinse with PBS for 5 min × 2 times; drop 100 μL of the secondary antibody and incubate at 37°C in an incubator for 30 min; rinse with PBS for 5 min × 2 times.

[0068] 8. DAB color development: Take the slides out of the incubator, rinse with PBS for 5 min × 2 times; drop 100 μL of the pre-prepared 0.05% DAB working solution for color development and incubate at room temperature for 1 - 15 min.

[0069] 9. Counterstaining: Wash the developed slides with water, then immerse them in 0.5% hematoxylin for counterstaining for 3 min, and wash with water to turn blue again.

[0070] 10. Dehydration: Dehydrate with alcohols at different concentrations (60%, 70%, 80%, 90%, 100%) for 5 min each.

[0071] 11. Clearing: Place the samples in xylene for 10 min after taking them out.

[0072] 12. Mounting: Mount the slides with neutral balsam and observe under a microscope.

[0073] 13. Take pictures of the results.

[0074] The results of the experimental group are as Figure 5 shown. The cell walls and nuclei of the sieve elements in the phloem in the middle part of the bamboo shoot body, including sieve mother cells, developing sieve elements, and companion cells, have no staining, which is consistent with the negative control. The cell walls and / or nuclei of the other cells except the phloem are brown, yellowish-brown, or brownish-black. The results of the control group are as Figure 6 shown. The nuclei of all types of cells are blue, and the cell walls have no obvious staining.

[0075] Comparative Example 1 1. Bamboo shoot bud collection: Use pruning shears commonly used in horticulture to cut off the bamboo shoot buds in the middle stage of development from the nodes of the underground stem, cut the bamboo shoot sheaths into small pieces of 0.5 cm × 0.5 cm, and perform tissue fixation.

[0076] 2. Dewaxing and rehydration of paraffin sections: Place the sections in xylene I for 20 min - xylene II for 20 min - absolute ethanol I for 10 min - absolute ethanol II for 10 min - 95% alcohol for 5 min - 90% alcohol for 5 min - 80% alcohol for 5 min - 70% alcohol for 5 min - distilled water wash in sequence.

[0077] 3. Safranin staining: Immerse the sections in 1% safranin staining solution for 2 h. Wash slightly with tap water to remove excess dye.

[0078] 4. Decolorization: Decolorize in 50%, 70%, and 80% gradient alcohols for 1 min each.

[0079] 5. Fast green staining: Immerse the sections in 0.5% fast green staining solution for 30 - 60 s. Decolorize in absolute ethanol for 30 s and in absolute ethanol for 1 min.

[0080] 6. Mounting: Dry the sections in an oven at 60 °C, then clear them in xylene for 5 min, and mount them with neutral balsam.

[0081] 7. Microscopic examination and image acquisition and analysis.

[0082] The results are as Figure 7As shown, the sieve elements are stained dark blue - green by safranin - fast green staining solution, and the morphology, size, and number of cell nuclei are not clear. The cell walls of other cells, although containing cellulose but in a lower content, are stained light blue.

[0083] Comparative Example 2 1. Bamboo shoot bud collection: Use pruning shears commonly used in horticulture to remove the bamboo shoot buds in the mid - development stage from the nodes of the underground stem. Cut the bamboo shoot body into small pieces of 0.5 cm×0.5 cm, and select the small pieces located in the upper part of the bamboo shoot body for tissue fixation.

[0084] 2. Tissue fixation, dehydration, clearing, infiltration with paraffin wax, embedding, sectioning, baking the sections, and dewaxing the sections to water.

[0085] 3. Antigen heat repair: Immerse the sections in the EDTA antigen repair solution, heat them in a microwave oven at medium power for 8 min, stop the heating for 5 min, then heat at low power for 5 min, and cool to room temperature. After cooling, wash with 0.01M PBS (pH 7.2 - 7.6) for 5 min×2 times.

[0086] 4. Add 3% H2O2 and incubate at room temperature for 10 min to inactivate endogenous enzymes. Rinse with PBS for 5 min×2 times.

[0087] 5. Serum blocking: Drop 5% goat serum on the sections and place them in a wet box at room temperature for 30 min. Wash with PBS for 5 min×2 times; 6. Incubate with the primary antibody: Drop PBS and incubate in a 4℃ refrigerator overnight.

[0088] 7. Incubate with the secondary antibody: Take the slides out of the refrigerator and warm them at 37℃ in an incubator for 30 min; rinse with PBS for 5 min×2 times; drop 100 μL of the secondary antibody and incubate at 37℃ in an incubator for 30 min; rinse with PBS for 5 min×2 times.

[0089] 8. DAB color development: Take the slides out of the incubator, rinse with PBS for 5 min×2 times; drop 100 μL of the pre - prepared 0.05% chromogenic agent DAB working solution and incubate at room temperature for 15 min.

[0090] 9. Counter - staining: Wash the slides after color development, then immerse them in 0.5% hematoxylin for counter - staining for 3 min, and wash with water to turn blue.

[0091] 10. Dehydration: Dehydrate with alcohols of different concentrations (60%, 70%, 80%, 90%, 100%) for 5 min for each concentration.

[0092] 11. Clearing: Place them in xylene for 10 min after taking them out.

[0093] 12. Sealing the slides: Seal the slides with neutral balsam and observe under a microscope.

[0094] 13. Take pictures of the results.

[0095] The results are as Figure 8 shown. Neither the phloem and xylem of the vascular tissue nor the cells of the ground tissue reacted with the antibody and were not stained, and the cell nuclei were blue.

[0096] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments based on these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A histological staining method for Phyllostachys spp., characterized in that: The following steps are involved: The tissue sections of the developing shoots were prepared as samples, and the sections were incubated with trimethylated histone antibodies, secondary antibodies and DAB color developing solution in sequence, and then counterstained with hematoxylin, dehydrated with alcohol and transparentized with xylene, and then sealed for microscopic observation.

2. The histological staining method according to claim 1, characterized in that: The bamboo shoot bud tissue comprises taking the bamboo shoot buds in the developmental stage from the nodes of the underground stems, and cutting the middle part of the bamboo shoot body or the bamboo shoot shell into pieces.

3. The histological staining method according to claim 2, characterized in that: The bamboo shoot tissue is sequentially subjected to tissue fixation, dehydration, transparency, wax immersion, embedding, slicing, baking and slicing dewaxing to water to prepare tissue slices.

4. The histological staining method according to claim 1, characterized in that: Prior to incubation with antibodies against trimethylated histones, antigen retrieval, inactivation of endogenous enzymes, and serum blocking were also performed.

5. The histological staining method according to claim 4, characterized in that: The antigen repair comprises placing the tissue section into an EDTA antigen repair solution for heat repair of the antigen, wherein the EDTA antigen repair solution comprises Tris-EDTA; The inactivation of endogenous enzymes comprises adding a 3% by volume H2O2 solution to the tissue slice; The serum blocking comprises dropping 5% by volume sheep serum into the tissue slices and then leaving the slices to stand at room temperature.

6. The histological staining method according to claim 1, characterized in that: The trimethylated histone antibody includes an antibody generated from trimethylated histone H3.

7. The histological staining method according to claim 1 or 6, characterized in that: The trimethylated histone antibody includes anti-H3K4me3.

8. The histological staining method according to claim 7, characterized in that: The dilution ratio of the anti-H3K4me3 is 1:50~200.

9. Use of the histological staining method according to any one of claims 1 to 8 in distinguishing phloem cells in plants of the genus Phyllostachys.

10. A method for distinguishing phloem cells of Phyllostachys spp., characterized in that: The method comprises staining a tissue section using the histological staining method according to any one of claims 1 to 8, and observing under a microscope that the early phloem cells are not stained, while the remaining cells are stained.

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