A method for histological staining using protein markers and its application in differentiating sieve elements and companion cells

Abstract

The present invention provides a method for histological staining using protein markers and its application in differentiating sieve elements and companion cells, belonging to the technical field of biological histology. The present invention discloses a method for differentiating early tubular cells of sieve elements and companion cells in plant phloem. By using epigenetic protein markers and histological staining, before the nuclei of sieve elements disappear in specific structures, two symbiotic tubular molecules in the phloem, namely sieve elements and companion cells, can be differentiated according to whether the cells are stained. At the same time, the specific location of the interaction between methylated histone and companion cells can be distinguished according to the staining location and depth, and the development degree of companion cells can be evaluated, with high sensitivity and high precision.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological histology, and particularly relates to a method for histological staining using protein markers and its application in differentiating sieve elements and companion cells. Background Art

[0002] Bamboo plants belong to the genus Phyllostachys of the Poaceae family. The vascular tissue is scattered in the ground tissue. The phloem of bamboo plants, like that of other plants, is composed of sieve elements and companion cells. Companion cells and sieve tubes are derived from the same mother cell and grow side by side, with equal lengths or the companion cells being slightly shorter than the sieve tubes. The cell walls of both sieve tubes and companion cells are not lignified and thickened. In addition, companion cells have abundant protoplasm.

[0003] For the shoot buds in the early and middle stages of development, various tubular cells are in a state of differentiation, and specific components and specialized structures are being formed. At this time, it is difficult to accurately distinguish them only by the size and shape of the cells. At the same time, the cell walls of sieve tubes and companion cells are not lignified and thickened. It is difficult to distinguish the sieve elements and companion cells in the phloem by conventional histological staining methods, such as safranin-fast green, etc.

[0004] Although it is possible to observe the ultrastructure of cells, including cell organelles, cytoplasm, and cell nuclei, etc., with the help of a transmission electron microscope (abbreviated as TEM), which provides more information for distinguishing tissue development and cell types; however, compared with the samples embedded in conventional paraffin, the TEM samples take a long time, have cumbersome steps, high operation difficulty, and high reagent costs, and are difficult to be widely applied. Summary of the Invention

[0005] The present invention provides a method for histological staining using protein markers and its application in differentiating sieve elements and companion cells, which can stain only companion cells by histological staining, thus meeting the easy differentiation under an electron microscope.

[0006] The present invention provides the application of an antibody in differentiating the early tubular cells of sieve elements and companion cells in plant phloem, and the antibody is an antibody generated using methylated histone.

[0007] In a preferred embodiment of the present invention, the methylated histone includes methylating histone H3.

[0008] The present invention also provides a method for differentiating the early tubular cells of sieve elements and companion cells in plant phloem, comprising the following steps: (1) taking a part of the tissue of the target plant and preparing tissue sections;

[0009] (2) immersing the tissue sections in an EDTA antigen retrieval solution to repair the antigen, then inactivating endogenous enzymes and performing blocking;

[0010] (3) Sequentially perform primary antibody incubation and secondary antibody incubation on the sealed tissue sections. The primary antibody is an antibody generated from methylated histone; the secondary antibody is HRP-labeled goat anti-rabbit IgG (Servicebio, GB23303, dilution ratio 1:200).

[0011] (4) Add a chromogenic agent to the tissue sections incubated in step (3) for color development, wash with water, then counterstain with hematoxylin, and wash with water to turn blue again.

[0012] (5) Dehydrate, clear, and mount the tissue sections that have turned blue again, and then observe under a microscope. The stained tubular cells are companion cells, and the unstained cells are sieve elements.

[0013] In a preferred embodiment of the present invention, the target plant in step (1) includes plants of the genus Phyllostachys.

[0014] In a preferred embodiment of the present invention, the primary antibody in step (3) includes anti-H3K27me3 antibody.

[0015] In a preferred embodiment of the present invention, when performing the primary antibody incubation, the dilution ratio is 1:50 - 200, and the incubation is carried out in an environment of 4°C for 8 - 12 h.

[0016] In a preferred embodiment of the present invention, before the secondary antibody incubation in step (3), it further includes rewarming to 37°C, rinsing with PBS, and then adding the secondary antibody dropwise.

[0017] The temperature for the secondary antibody incubation is 37°C, and the time is 30 min.

[0018] In a preferred embodiment of the present invention, the chromogenic agent in step (4) includes DAB, and the working solution concentration of DAB is 0.05% (w / v), and the incubation is carried out at room temperature (25°C) for 1 - 15 min.

[0019] In a preferred embodiment of the present invention, the dehydration in step (5) includes dehydration using alcohol solutions with gradient concentrations.

[0020] In a preferred embodiment of the present invention, the clearing in step (5) includes soaking in xylene solution for 10 min.

[0021] The present invention also provides the application of the above method in determining the cell types of phloem tubular elements and / or evaluating the developmental stages.

[0022] The present invention also provides the application of the above method in detecting the localization, quantity, and distribution of H3K27me3 in plant tissues.

[0023] Beneficial effects: The present invention discloses a method for distinguishing phloem sieve elements and early companion cell tubular cells in plants. By using epigenetic protein markers and histological staining methods, before the nuclei of sieve elements, which are specific structures, disappear, two symbiotic tubular molecules in the phloem, namely sieve elements and companion cells, can be distinguished based on whether the cells are stained. At the same time, the specific location of the interaction between methylated histone and companion cells can be distinguished according to the staining position and depth, and the developmental degree of companion cells can be evaluated, with high sensitivity and high precision.

[0024] In the embodiments of the present invention, histological staining is performed using an antibody against trimethylated lysine 27 of histone H3 (H3K27me3). Taking tissue sections as samples, according to the staining situation, the cell types of phloem tubular molecules can be specifically distinguished under an optical microscope. For example, companion cells can exhibit staining based on the expression quantity of antibody H3K27me3 in the tissue, such as showing brown, yellow-brown, or brownish-tan colors, while sieve elements do not show obvious staining. At the same time, by using the method of the present invention, with the aid of an electron microscope, the expression of H3K27me3 protein in tissues or cells can be detected to determine cell types, evaluate developmental stages, and detect the localization, quantity, and distribution of H3K27me3 in tissues. The method of the present invention is simple, easy to implement, low in cost, and has high detection sensitivity and high precision. Description of the Drawings

[0025] Figure 1 Microscopic examination diagram of the staining result of Example 1 (scale bar: 50 μm);

[0026] Figure 2 Microscopic examination diagram of the staining result of the control group of Example 1 (scale bar: 50 μm);

[0027] Figure 3 Microscopic examination diagram of the staining result of Example 2 (scale bar: 50 μm);

[0028] Figure 4 Microscopic examination diagram of the staining result of the control group of Example 2 (scale bar: 20 μm);

[0029] Figure 5 Microscopic examination diagram of the staining result of Example 3 (scale bar: 50 μm);

[0030] Figure 6 Microscopic examination diagram of the staining result of the control group of Example 3 (scale bar: 50 μm);

[0031] Figure 7 Microscopic examination diagram of the staining result of Comparative Example 1 (scale bar: 50 μm);

[0032] Figure 8 Microscopic examination diagram of the staining result of Comparative Example 2 (scale bar: 100 μm). Detailed Embodiments

[0033] The present invention provides the application of an antibody in differentiating phloem sieve elements and early companion cell tubular cells of plants, and the antibody is generated using methylated histone.

[0034] The histone of the present invention includes histone H3, and the methylated histone includes trimethylation on key amino acids of histone H3, such as trimethylation on the 27th amino acid of histone H3 used in the examples, abbreviated as H3K27me3. The antibody of the present invention is a polyclonal antibody or a monoclonal antibody generated using the methylated histone. The antibody used in the examples is a rabbit polyclonal antibody purchased from Abbkine Scientific Co., Ltd., and its product number is A22396.

[0035] The differentiation of the present invention includes performing histological staining on plant tissues using the antibody, and then differentiating under an optical microscope. Among them, the stained ones are companion cells, and the ones not significantly stained are sieve elements.

[0036] The present invention also provides a method for differentiating phloem sieve elements and early companion cell tubular cells of plants, including the following steps: (1) After removing a part of the tissue of the target plant, successively perform fixation, dehydration, clearing, infiltration with paraffin wax, embedding, sectioning, baking the sections, and dewaxing the sections to water to prepare tissue sections;

[0037] (2) Immerse the tissue sections in an EDTA antigen retrieval solution to retrieve the antigen, and then inactivate endogenous enzymes and perform blocking;

[0038] (3) Perform primary antibody incubation and secondary antibody incubation on the blocked tissue sections in sequence. The primary antibody is a polyclonal antibody generated using methylated histone; the secondary antibody is HRP-labeled goat anti-rabbit IgG;

[0039] (4) Drop a chromogenic agent onto the tissue sections incubated in step (3) for chromogenesis, wash with water, and then counterstain with hematoxylin, and wash with water to turn blue;

[0040] (5) Dehydrate, clear, and mount the tissue sections that have turned blue, and then observe under a microscope. The stained tubular cells are companion cells, and the unstained cells are sieve elements.

[0041] The plant described in the present invention can be a plant of the genus Phyllostachys, such as bamboo. During the development period of the bamboo shoot bud, including the early, middle, and late stages of development, in the phloem at this time, both the sieve tubes and companion cells are tubular structures, and the cell walls are not thickened by lignification, so it is difficult to distinguish them. The present invention can distinguish the companion cells and sieve elements in the phloem of bamboo shoot buds. When making such a distinction, first, it is necessary to collect bamboo shoot buds. For example, use pruning shears to cut off the developing bamboo shoot buds from the nodes of the underground stem, cut the middle part of the bamboo shoot body into small pieces of 0.5 cm × 0.5 cm, and perform tissue fixation, dehydration, clearing, infiltration with paraffin, embedding, sectioning, baking, and dewaxing the sections to water to obtain tissue sections. The present invention does not specifically describe the specific preparation method of the tissue sections, and it can be prepared by conventional means in the art, such as the methods described in the references (Zhang Qiaoying, Yao Genyou. Preparation methods and techniques of paraffin tissue microarrays. Journal of Practical Oncology, 2005 (06), 551 - 553; You Peilei, Xu Jie, Qian Zhongying. Callus induction and section observation of Abelmoschus esculentus. Acta Agriculturae Shanghai, 2007, (03): 92 - 95).

[0042] The present invention repairs the antigens of the tissue sections. During the specific operation, immerse the tissue sections in the EDTA antigen repair solution, heat them in a microwave oven at medium heat (60 °C) for 8 min, stop heating for 5 min, then heat at low heat (30 °C) for 5 min, and cool to room temperature. The composition of the EDTA antigen repair solution described in the present invention includes Tris - EDTA. Mix 10 mL of 20×Tris - EDTA (pH 8.0) with 190 mL of distilled water to obtain a 1× antigen repair solution with 1 mM EDTA at pH 8.0. 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), wash for 5 min each time, and wash a total of 2 times.

[0043] After the above washing, the present invention performs the operation of inactivating endogenous enzymes. For example, add an H2O2 aqueous solution to the above tissue sections and let it stand at room temperature for a period of time. In one embodiment, add an H2O2 aqueous solution with a volume percentage of 3% and let it stand at room temperature for 10 min; then wash with the above PBS again, 5 min each time, and wash a total of 2 times.

[0044] The present invention blocks the tissue sections after enzyme inactivation. The blocking solution can be serum. For example, 5% goat serum is used in the embodiment. During the specific operation, add 5% goat serum dropwise to the sections and place them in a wet box at room temperature for 30 min; then wash with 0.01 M PBS (pH 7.2 - 7.6), wash for 5 min each time, and wash a total of 2 times.

[0045] In the present invention, a primary antibody is added dropwise to the section after being sealed and incubated. The primary antibody is a polyclonal antibody generated from methylated histone, such as the rabbit polyclonal antibody anti-H3K27me3 used in the examples, and the dilution ratio is 1:50 to 200, such as 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 adding the diluted primary antibody dropwise in the present invention, incubation is required. The temperature of the incubation is 4°C and the time is 8 to 12 hours. In the examples, after adding the primary antibody, it is placed in a 4°C refrigerator overnight.

[0046] In the present invention, the slide after incubating with the primary antibody is taken out and rewarmed in an incubator at 37°C for 30 min; it is rinsed with PBS, 5 min each time, and rinsed 2 times in total; then secondary antibody incubation is carried out. 50 to 100 μL of the secondary antibody is added dropwise to the slide and incubated in an incubator at 37°C for 30 min. After the incubation, it is rinsed with PBS again, 5 min each time, and rinsed 2 times in total. The secondary antibody used in the present invention is HRP-labeled goat anti-rabbit IgG (Servicebio, GB23303, and the dilution ratio is 1:200.

[0047] In the present invention, DAB color development is carried out on the slide after incubating with the secondary antibody. 50 to 100 μL of the pre-prepared color developer DAB working solution (0.05%) is added dropwise to the slide and incubated at room temperature for 1 to 15 min, and the reaction time is controlled according to the color development.

[0048] After the DAB color development in the present invention, counterstaining is required, that is, the slide after DAB color development is first washed with water, and then immersed in hematoxylin (0.5% w / v) for counterstaining for 1 to 3 min, and then washed with water to turn blue.

[0049] In the present invention, dehydration is carried out after the counterstaining. The dehydration is carried out step by step using alcohol 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 to 100%, and each concentration can be 60%, 70%, 80%, 90% and 100%, and each stage of dehydration is 5 min.

[0050] In the present invention, the slide after dehydration is subjected to a clearing operation, such as being placed in xylene for 10 min, and then sealed with neutral balsam, and then it can be observed and photographed under an optical microscope.

[0051] After staining by the method of the present invention, the companion cells in the phloem are colored, and the color depends on the number of antibodies H3K27me3 expressed in the tissue, such as showing yellow, brown, yellow-brown or brownish-tan. There is no obvious lignin deposition in the sieve elements of the phloem, so there is no obvious staining.

[0052] The present invention also provides an application of the above method in the evaluation of cell types and / or developmental stages of phloem sieve elements.

[0053] Plant phloem is an important part of the vascular tissue and is responsible for the long-distance transport of organic substances. Sieve elements and companion cells are two long tubular cells in the phloem. In the upper and middle parts of the bamboo shoots in the early stage of development, the sieve element cells are small, with large nuclei and dense cytoplasm, showing typical meristematic characteristics. In the upper and middle parts of the bamboo shoots in the middle stage of development, the sieve element cells elongate longitudinally. In the upper and middle parts of the bamboo shoots in the late stage of development, during the organelle degradation stage, sieve plates are formed (sieve tube elements), that is, plasmodesmata are enlarged into sieve pores, and callose is deposited around them.

[0054] The present invention also provides an application of the above method in detecting the localization, quantity, and distribution of H3K27me3 in plant tissues.

[0055] H3K27me3 (trimethylation of lysine 27 of histone H3) is an important epigenetic marker in plants. It plays a key role in gene silencing by inhibiting the transcription of target genes (such as developmental regulatory genes and stress response genes) through chromatin compaction; it also maintains the pluripotency of stem cells in meristems or specific cells. In addition, it is also involved in epigenetic regulation under biotic stresses (such as drought and low temperature). The differentiation and maturation of phloem sieve elements and companion cells are the results of selective transcription and expression of genes, and at the same time, it is also an adaptive developmental mechanism for plants to respond to external environmental conditions. Through the above technology, the epigenetic regulation patterns of H3K27me3 in bamboo shoot tissues and cells can be accurately analyzed by observing the location of antibody expression, the intensity of expression, that is, the depth of staining, laying a foundation for the research of gene silencing and developmental mechanisms.

[0056] To further illustrate the present invention, the following examples will describe in detail a method for histological staining using protein markers and its application in differentiating sieve elements and companion cells provided by the present invention, but they should not be construed as limiting the protection scope of the present invention.

[0057] The room temperature referred to in the present invention is 25°C.

[0058] Example 1

[0059] 1. Collection of bamboo shoots: Use pruning shears commonly used in horticulture to cut the bamboo shoots in the early stage of development 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 in the upper and middle parts of the bamboo shoot body for tissue fixation.

[0060] 2. Tissue fixation, dehydration, clearing, infiltration with paraffin wax, embedding, sectioning, baking the sections, and dewaxing the sections to water.

[0061] 3. Heat repair antigen: Immerse the sections in EDTA antigen repair solution, heat 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 0.01M PBS (pH 7.2 - 7.6) for 5 min × 2 times.

[0062] 4. Add 3% H2O2 and incubate at room temperature for 10 min to inactivate endogenous enzymes. Rinse with PBS for 5 min × 2 times.

[0063] 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.

[0064] 6. Incubate with primary antibody: Drop appropriately diluted primary antibody anti-H3K27me3 (A22396, 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.

[0065] 7. Incubate with 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 50 - 100 μL of secondary antibody and incubate at 37°C in an incubator for 30 min; rinse with PBS for 5 min × 2 times.

[0066] 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 DAB working solution for color development and incubate at room temperature for 10 min, and control the reaction time according to the color development.

[0067] 9. Counterstain: Wash the slides after color development, then soak them in hematoxylin for counterstaining for 1 - 3 min, and wash with water to turn blue.

[0068] 10. Dehydration: Gradually dehydrate with 60%, 70%, 80%, 90% and 100% alcohol in sequence, 5 min for each step.

[0069] 11. Clearing: Place in xylene for 10 min after taking out.

[0070] 12. Sealing: Seal with neutral balsam and observe under a microscope.

[0071] 13. Take pictures of the results.

[0072] The results of the experimental group are as Figure 1 shown. The companion cells in the phloem are stained yellowish-brown, while the sieve tubes symbiotic with them do not react with the antibody and are not stained; the results of the control group are as Figure 2 shown. Neither the companion cells in the phloem nor the sieve tubes symbiotic with them react with the antibody, and the cell nuclei are all blue, with no obvious difference.

[0073] Example 2

[0074] 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 in the upper and middle parts of the bamboo shoot body for tissue fixation.

[0075] 2. Tissue fixation, dehydration, clearing, infiltration with paraffin wax, embedding, sectioning, baking the sections, and dewaxing the sections to water.

[0076] 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, 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.

[0077] 4. Add 3% H2O2 and incubate at room temperature for 10 min to inactivate endogenous enzymes. Rinse with PBS for 5 min × 2 times.

[0078] 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.

[0079] 6. Incubate with the primary antibody: Drop the appropriately diluted primary antibody anti-H3K27me3 (A22396, rabbit polyclonal antibody, Abbkine Scientific Co., Ltd., Wuhan, China) (dilution ratio 1:100) on the experimental group sections, and drop an equal amount of PBS on the control group sections, and incubate overnight in a 4°C refrigerator.

[0080] 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 50 - 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.

[0081] 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 DAB working solution for color development and incubate at room temperature for 10 min, and control the reaction time according to the color development.

[0082] 9. Counterstaining: Wash the slides after color development, then soak them in hematoxylin for counterstaining for 1 - 3 min, and wash with water to turn blue.

[0083] 10. Dehydration: Gradually dehydrate with 60%, 70%, 80%, 90% and 100% alcohol in sequence, 5 min for each stage.

[0084] 11. Clearing: Place them in xylene for 10 min after taking out.

[0085] 12. Sealing the slides: Seal the slides with neutral balsam and observe under a microscope.

[0086] 13. Photograph the results.

[0087] The results of the experimental group are as Figure 3 shown. The companion cells in the phloem were stained yellowish-brown, while the sieve tubes symbiotic with them did not react with the antibody and were not stained; the results of the control group are as Figure 4 shown. Neither the companion cells in the phloem nor the sieve tubes symbiotic with them reacted with the antibody, and the cell nuclei were all blue, showing no obvious difference.

[0088] Example 3

[0089] 1. Shoot bud collection: Use pruning shears commonly used in horticulture to remove the shoot buds in the late development stage 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 in the upper-middle part of the shoot body for tissue fixation.

[0090] 2. Tissue fixation, dehydration, clearing, infiltration with paraffin wax, embedding, sectioning, baking the sections, and dewaxing the sections to water.

[0091] 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 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.

[0092] 4. Add 3% H2O2 and incubate at room temperature for 10 min to inactivate endogenous enzymes. Rinse with PBS for 5 min × 2 times.

[0093] 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.

[0094] 6. Incubate with the primary antibody: Drop the appropriately diluted primary antibody anti-H3K27me3 (A22396, 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.

[0095] 7. Incubate with the secondary antibody: Take the slides out of the refrigerator and rewarm them in an incubator at 37°C for 30 min; rinse with PBS for 5 min × 2 times; drop 50 - 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.

[0096] 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 DAB working solution for color development and incubate at room temperature for 10 min, and control the reaction time according to the color development.

[0097] 9. Counterstaining: Wash the developed slides with water, then immerse them in hematoxylin for counterstaining for 1 - 3 min, and wash with water to turn blue again.

[0098] 10. Dehydration: Gradually dehydrate with 60%, 70%, 80%, 90% and 100% alcohol in sequence, 5 min for each step.

[0099] 11. Clearing: Place it in xylene for 10 min after taking out.

[0100] 12. Mounting: Mount with neutral balsam and observe under a microscope.

[0101] 13. Take pictures of the results.

[0102] The results of the experimental group are as Figure 5 shown. The companion cells in the phloem are stained yellowish-brown, while the sieve tubes symbiotic with them do not react with the antibody and are not stained; the results of the control group are as Figure 6 shown. Neither the companion cells in the phloem nor the sieve tubes symbiotic with them react with the antibody, and the cell nuclei are all blue, showing no obvious difference.

[0103] Comparative Example 1

[0104] 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 perform tissue fixation.

[0105] 2. Dewaxing the paraffin section to water: Put the section into 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.

[0106] 3. Safranin staining: Immerse the section in 1% safranin staining solution for 1 - 2 h. Wash slightly with tap water to wash away the excess dye.

[0107] 4. Decolorization: Decolorize in 50%, 70%, 80% gradient alcohol for 1 min each.

[0108] 5. Fast green staining: Immerse the section 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.

[0109] 6. Mounting: Bake the section in an oven at 60 °C until dry, then clear in xylene for 5 min and mount with neutral balsam.

[0110] 7. Microscopic examination and image acquisition and analysis.

[0111] The results are as Figure 7As shown, both the sieve tubes and companion cells in the phloem are stained green by safranin-fast green, making it difficult to distinguish between the two by color.

[0112] Comparative Example 2

[0113] 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 for tissue fixation.

[0114] 2. Tissue fixation, dehydration, clearing, infiltration with paraffin wax, embedding, sectioning, baking the sections, and dewaxing the sections to water.

[0115] 3. Heat antigen repair: Immerse the sections in the EDTA antigen repair solution and heat them in a microwave oven at medium power for 8 minutes, stop heating for 5 minutes, then heat at low power for 5 minutes, and cool to room temperature. After cooling, wash with 0.01M PBS (pH 7.2 - 7.6) for 5 minutes × 2 times.

[0116] 4. Add 3% H2O2 and inactivate endogenous enzymes at room temperature for 10 minutes. Rinse with PBS for 5 minutes × 2 times.

[0117] 5. Serum blocking: Drop 5% sheep serum on the sections and place them in a wet box at room temperature for 30 minutes. Wash with PBS for 5 minutes × 2 times;

[0118] 6. Incubation: Drop PBS and incubate overnight in a 4°C refrigerator.

[0119] 7. Incubate with secondary antibody: Take the slides out of the refrigerator and warm them at 37°C in an incubator for 30 minutes; rinse with PBS for 5 minutes × 2 times; drop 50 - 100 μL of secondary antibody and incubate at 37°C in an incubator for 30 minutes; rinse with PBS for 5 minutes × 2 times.

[0120] 8. DAB color development: Take the slides out of the incubator and rinse with PBS for 5 minutes × 2 times; drop 50 - 100 μL of the pre-prepared DAB working solution for color development and incubate at room temperature for 1 - 15 minutes, and control the reaction time according to the color development.

[0121] 9. Counterstaining: Wash the slides after color development, then soak them in hematoxylin for counterstaining for 1 - 3 minutes, and wash with water to turn blue.

[0122] 10. Dehydration: Dehydrate with alcohols of various concentrations (60 - 100%), 5 minutes for each concentration.

[0123] 11. Clearing: Place them in xylene for 10 minutes after taking them out.

[0124] 12. Sealing the slides: Seal the slides with neutral balsam and observe under a microscope.

[0125] 13. Take pictures of the results.

[0126] The results are as Figure 8 shown. Neither the sieve elements nor the companion cells in the phloem reacted with the antibody and were not stained.

[0127] 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 method for differentiating the sieve elements and early tubular cells of companion cells in the phloem of Phyllostachys plants, characterized in that It includes the following steps: (1) Take a part of the tissue of the target plant to prepare a tissue section; (2) Immerse the tissue section in an EDTA antigen repair solution to repair the antigen, and then inactivate the endogenous enzyme and then block it; the inactivation of the endogenous enzyme includes adding an H2O2 aqueous solution to the tissue section after antigen repair and allowing it to stand at room temperature for a period of time; (3) Perform primary antibody incubation and secondary antibody incubation on the blocked tissue section in sequence. The primary antibody is an H3K27me3 antibody; the secondary antibody is HRP-labeled goat anti-rabbit IgG; (4) Drop DAB on the tissue section incubated in step (3) for color development, wash with water, and then counterstain with hematoxylin, and wash with water to turn blue; (5) Dehydrate, clear and mount the tissue section that has turned blue, and then observe it under a microscope. The stained tubular cells are companion cells, and the unstained cells are sieve elements.

2. The method according to claim 1, wherein When performing the primary antibody incubation, the dilution ratio is 1:50 - 200, and the incubation is carried out in an environment at 4°C for 8 - 12 h.

3. The method according to claim 1, wherein Before the secondary antibody incubation in step (3), it also includes rewarming to 37°C, rinsing with PBS, and then dropping the secondary antibody; The temperature of the secondary antibody incubation is 37°C, and the time of the secondary antibody incubation is 30 min.

4. The method according to claim 1, wherein The working solution concentration of DAB in step (4) is 0.05% (w / v), and the incubation is carried out at room temperature for 1 - 15 min.

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