A histological staining method for Phyllostachys genus and a method for distinguishing phloem cells

Abstract

The present invention provides a histological staining method for plants of the genus Phyllostachys and a method for distinguishing phloem cells, and belongs to the technical field of tissue sections. The present invention provides a histological staining method for plants of the genus Phyllostachys, which uses antibodies generated by trimethylated histones for histological staining, and uses tissue sections as samples. The antibodies are expressed and undergo color development in cells other than the phloem, but are not expressed in the phloem, resulting in no coloring. Therefore, the method of the present invention can perform specific staining on different tissue cells, has high sensitivity, and is low in cost. An ordinary electron microscope can be used to determine whether the cell is a phloem cell based on whether the cell wall and / or cell nucleus are colored, and because the cell wall of the phloem is not colored, the cell nucleus is clearly visible. The histological staining method of the present invention is particularly suitable for detecting the morphological characteristics of the cell nuclei of early phloem cells.

Description

Technical Field

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

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

[0003] Currently, for bamboo and most plants, qualitative histological staining methods are used to distinguish them by comparing differences in cell wall color. For example, after staining with Safranin Fast Green, the cell walls of the sieve elements in the phloem appear dark blue, while the thickened cell walls of the xylem appear brick red. However, conventional staining results in the staining of all cell walls, as the main components of the cell walls of various tissues are essentially the same, but the degree of staining varies, resulting in poor specificity and low sensitivity. Furthermore, due to the staining of the cell walls, the size, morphology, and distribution of the nuclei within the phloem cells are difficult to observe.

[0004] In addition, although the ultrastructure of cells, including organelles, cytoplasm and nucleus, can be observed with the help of a transmission electron microscope (TEM), providing more information for distinguishing tissue development and cell types; however, compared with conventional paraffin-embedded samples, TEM samples take a long time, have complicated steps, and are difficult to operate; the reagent costs are high; and TEM equipment is expensive and has not yet been popularized. Summary of the Invention

[0005] The present invention provides a histological staining method for Phyllostachys spp. and a method for distinguishing phloem cells. The method can specifically stain tissues, distinguish phloem cells by color under a common electron microscope, and clearly observe the internal structural characteristics of the phloem.

[0006] The invention provides a histological staining method for Phyllostachys genus plants, comprising the following steps: preparing bamboo shoot bud tissue in a developmental stage into tissue sections, using the tissue sections as samples, sequentially incubating with an antibody against trimethylated histones, a secondary antibody, and a DAB color developing solution, then counter-staining with hematoxylin, dehydrating with alcohol, and transparentizing with xylene, then sealing the sections for microscopic observation.

[0007] In a preferred embodiment of the present invention, the bamboo shoot bud tissue comprises removing 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.

[0008] In a preferred embodiment of the present invention, the bamboo shoot tissue is sequentially fixed, dehydrated, transparent, immersed in wax, embedded, sliced, baked and dewaxed to water to prepare tissue sections.

[0009] In a preferred embodiment of the present invention, before incubation with an antibody against trimethylated histones, the method further includes antigen repair, inactivation of endogenous enzymes and serum blocking.

[0010] In a preferred embodiment of the present invention, the antigen repair comprises placing the tissue section into an EDTA antigen repair solution for heat repair of the antigen, wherein the composition of the EDTA antigen repair solution is Tris-EDTA;

[0011] The inactivation of endogenous enzymes comprises adding a 3% by volume H2O2 solution to the tissue slice;

[0012] The serum blocking comprises adding 5% by volume sheep serum to the tissue slices and then leaving the slices to stand at room temperature.

[0013] In a preferred embodiment of the present invention, the antibody against trimethylated histone includes an antibody generated against trimethylated histone H3.

[0014] In a preferred embodiment of the present invention, the antibody against trimethylated histones includes anti-H3K4me3.

[0015] In a preferred embodiment of the present invention, the dilution ratio of the anti-H3K4me3 is 1:50-200.

[0016] The present invention also provides application of the histological staining method in distinguishing phloem cells in plants of the genus Phyllostachys.

[0017] The present invention also provides a method for distinguishing phloem cells of Phyllostachys genus plants, comprising staining tissue sections using the above-mentioned histological staining method, and observing under a microscope, wherein the early phloem cells are not stained, while the remaining cells are stained.

[0018] Beneficial effects: The present invention provides a histological staining method for plants of the genus Phyllostachys, which uses antibodies generated by trimethylated histones for histological staining. Tissue sections are used as samples, and the antibodies are expressed in cells other than the phloem, and color reactions occur in all cells. The degree of staining depends on the amount of the antibodies expressed in the tissue, and in the cell walls and / or cell nuclei, brown, yellow-brown or tan appears. However, the antibodies are not expressed in the phloem, and there is no staining. Therefore, the method of the present invention can be used to specifically stain different tissue cells, with high sensitivity and low cost. An ordinary electron microscope can be used to determine whether the cell is a phloem cell based on whether the cell wall and / or cell nucleus is stained. Moreover, since the cell wall of the phloem is not stained, the cell nucleus is clearly visible. The histological staining method of the present invention is particularly suitable for detecting the morphological characteristics of the nuclei of early phloem cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The microscopic examination image of the staining result of Example 1 (scale 50 μm);

[0020] Figure 2 This is a microscopic examination of the staining results of the control group in Example 1 (scale 50 μm);

[0021] Figure 3 The microscopic examination image of the staining result of Example 2 (scale 50 μm);

[0022] Figure 4 This is a microscopic examination of the staining results of the control group in Example 2 (scale 50 μm);

[0023] Figure 5 The microscopic examination image of the staining result of Example 3 (scale 50 μm);

[0024] Figure 6 This is a microscopic examination of the staining results of the control group in Example 3 (scale 50 μm);

[0025] Figure 7 The microscopic examination image of the staining result of Comparative Example 1 (scale 50 μm);

[0026] Figure 8 This is a microscopic examination of the staining results of Comparative Example 2 (scale 100 μm). DETAILED DESCRIPTION

[0027] The invention provides a histological staining method for Phyllostachys genus plants, comprising the following steps: preparing bamboo shoot bud tissue in a developmental stage into tissue sections, using the tissue sections as samples, sequentially incubating with an antibody against trimethylated histones, a secondary antibody, and a DAB color developing solution, then counter-staining with hematoxylin, dehydrating with alcohol, and transparentizing with xylene, then sealing the sections for microscopic observation.

[0028] The present invention uses tissue slices as samples, and the method for preparing the tissue slices is not particularly limited. For example, the selected tissue is sequentially fixed, dehydrated, transparent, wax-impregnated, embedded, sliced, baked, and dewaxed to water to prepare the tissue slices. When preparing the tissue slices, reference can be made to methods in the literature, such as the method of You Peilei (You Peilei, Xu Jie, Qian Zhongying. Production of Okra Callus and Observation of Sections. Shanghai Journal of Agriculture, 2007, (03): 92-95) or Zhang Qiaoying (Zhang Qiaoying, Yao Genyou. Method and Techniques for Preparing Paraffin Tissue Microarrays. Practical Journal of Oncology, 2005 (06), 551-553).

[0029] In the examples of the present invention, the histological staining was performed using bamboo as an example, wherein the tissue sections were prepared using bamboo shoot tissue. The bamboo shoot tissue was prepared by removing bamboo shoots from the nodes of the underground stem during development and cutting the middle portion of the shoot into pieces, or cutting the bamboo shoot shell into pieces. The developmental stages described herein include early development, mid-development, and late development. In the upper and middle portions of bamboo shoots in the early developmental stage, the cells that make up the vascular tissue are elongated to varying degrees. At this point, the procambium has differentiated into the protoxylem and is strongly lignified, while the phloem is not significantly specialized. In the upper and middle portions of bamboo shoots in the mid-developmental stage, the end walls of the sieve elements are significantly specialized. In the upper and middle portions of bamboo shoots in the late developmental stage, the sieve elements and companion cells of the phloem are significantly specialized. Compared to the vascular tissue in the internodes adjacent to the node, the vascular tissue cells in the culm sheath differentiate earlier.

[0030] After the corresponding tissue sections are prepared, the sections need to be repaired with antigens, inactivated with endogenous enzymes, and blocked with serum before they can be incubated with primary and secondary antibodies.

[0031] The present invention performs antigen retrieval on the tissue sections. Specifically, the sections are immersed in EDTA antigen retrieval solution, heated in a microwave oven at medium heat (60°C) for 8 minutes, then removed from the heat for 5 minutes, and then heated at low heat (30°C) for 5 minutes before cooling to room temperature. The EDTA antigen retrieval solution comprises Tris-EDTA. To prepare the solution, 10 mL of 20× Tris-EDTA (pH 8.0) is mixed with 190 mL of distilled water to obtain a 1× antigen retrieval solution containing 1 mM EDTA at pH 8.0. After cooling to room temperature, the sections are then washed twice with 0.01M PBS (pH 7.2-7.6), each wash lasting 5 minutes.

[0032] The present invention performs an operation to extinguish endogenous enzymes after the above-mentioned washing, such as adding H2O2 solution to the above-mentioned tissue slices and letting them stand at room temperature for a period of time. In one embodiment, a 3% H2O2 solution by volume is added and let stand at room temperature for 10 minutes; then the above-mentioned PBS is used for washing, each time for 5 minutes, for a total of 2 washes.

[0033] The present invention blocks the tissue sections after enzyme inactivation. Serum can be used for blocking. For example, 5% sheep serum is used in the embodiment. In the specific operation, 5% sheep serum is added dropwise to the sections and placed in a wet box at room temperature for 30 minutes. The sections are then washed with 0.01M PBS (pH 7.2-7.6), each wash lasting 5 minutes, for a total of 2 washes.

[0034] In the present invention, a primary antibody is added dropwise to the blocked slice for incubation. The primary antibody is a polyclonal antibody generated from trimethylated histones. The antibody generated from trimethylated histone H3 used in the examples, such as anti-H3K4me3, is a rabbit polyclonal antibody purchased from Abotek Biotechnology Co., Ltd., with the product number A22225. When the primary antibody is added dropwise, the primary antibody needs to be diluted at a dilution ratio of 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. In the present invention, after the appropriately diluted primary antibody is added dropwise, incubation is required. The incubation temperature is 4° C. and the time is 8 to 12 hours. In the embodiment, after the primary antibody is added dropwise, the mixture is placed in a 4° C. refrigerator overnight.

[0035] After primary antibody incubation, the slides were removed and rewarmed in a 37°C incubator for 30 minutes. The slides were then rinsed with PBS for 5 minutes each time, for a total of two rinses. Secondary antibody incubation was then performed by adding 50–100 μL of secondary antibody to the slides and incubating them in a 37°C incubator for 30 minutes. Following this incubation, the slides were rinsed with PBS for 5 minutes each time, for a total of two rinses. The secondary antibody used in the present examples was HRP-labeled goat anti-rabbit IgG (Servicebio, GB23303, diluted 1:200).

[0036] The present invention performs DAB color development on the glass slide after incubation with the secondary antibody, drops 50-100 μL of a pre-prepared color developer DAB working solution (0.05%, w / v) onto the glass slide, and incubates at room temperature for 1-15 minutes, controlling the reaction time according to the color development.

[0037] In the present invention, after the DAB color development, the slide needs to be restained. That is, the slide after DAB color development is first washed with water, then immersed in 0.5% w / v hematoxylin for re-staining for 1 to 3 minutes, and then washed with water to return to blue.

[0038] The present invention performs dehydration after the redyeing, and the dehydration uses alcohol with gradient concentrations for step-by-step dehydration, and the volume percentage of the alcohol solution used is increased successively to achieve the dehydration purpose. The volume percentage of the alcohol solution used is 60-100%, and the concentration of each level can be 60%, 70%, 80%, 90% and 100%, and each level of dehydration takes 5 minutes.

[0039] The present invention performs a transparent operation on the dehydrated slide, such as placing it in xylene for 10 minutes, and then sealing the slide with neutral gum, and then the slide can be observed and photographed under an electron microscope.

[0040] The present invention also provides application of the histological staining method in distinguishing phloem cells in plants of the genus Phyllostachys.

[0041] When specialized structures of phloem cells such as sieve elements, such as sieve plates, have not yet formed or are in the process of formation, the present invention utilizes the fact that phloem cells are not stained by the antibodies to distinguish unstained early phloem cells (including sieve mother cells, developing sieve elements and companion cells) from other stained tissues, including xylem, epidermis and basic tissues composed of parenchyma cells; 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 can all be clearly observed with the aid of a conventional electron microscope.

[0042] The present invention also provides a method for distinguishing phloem cells of Phyllostachys genus plants, comprising staining tissue sections using the above-mentioned histological staining method, and observing under a microscope, wherein the early phloem cells are not stained, while the remaining cells are stained.

[0043] When distinguished by the method of the present invention, the cell walls and nuclei of the tubular elements of the phloem, including sieve elements and companion cells, are not stained, while the cell walls and / or nuclei of cells other than the phloem are brown, yellow-brown or tan.

[0044] To further illustrate the present invention, a histological staining method for Phyllostachys spp. and a method for distinguishing phloem cells provided by the present invention are described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0045] Example 1

[0046] 1. Collection of bamboo shoots: Use common gardening shears to remove the early-stage bamboo shoots from the nodes of the underground stems, cut the bamboo shoots into small pieces of 0.5cm×0.5cm, and select the small pieces located at the top of the bamboo shoots for tissue fixation.

[0047] 2. Tissue fixation, dehydration, transparency, wax immersion, embedding, sectioning, baking, and dewaxing of sections until they are hydrated.

[0048] 3. Heat antigen retrieval: Immerse sections in EDTA antigen retrieval solution and microwave on medium heat for 8 minutes, then off heat for 5 minutes, then on low heat for 5 minutes. Cool to room temperature. After cooling, wash twice in 0.01M PBS (pH 7.2-7.6) for 5 minutes each.

[0049] 4. Add 3% H2O2 and incubate at room temperature for 10 minutes to inactivate endogenous enzymes. Rinse with PBS for 5 minutes twice.

[0050] 5. Serum blocking: Add 5% goat serum to the sections and place in a wet chamber at room temperature for 30 minutes. Wash with PBS for 5 minutes twice.

[0051] 6. Incubation with primary antibody: Add appropriately diluted primary antibody anti-H3K4me3 (A22225, rabbit polyclonal antibody, Ibotek Biotechnology Co., Ltd., Wuhan, China) (dilution ratio 1:100). Add an equal amount of PBS to the control group and incubate at 4°C overnight.

[0052] 7. Incubation with secondary antibody: Remove the slides from the refrigerator and rewarm them in a 37°C incubator for 30 min; rinse with PBS for 5 min × 2 times; add 100 μL of secondary antibody and incubate at 37°C for 30 min; rinse with PBS for 5 min × 2 times.

[0053] 8. DAB color development: Remove the slide from the incubator and rinse with PBS for 5 min × 2 times; add 50-100 μL of pre-prepared 0.05% color developer DAB working solution and incubate at room temperature for 15 min.

[0054] 9. Restaining: Wash the slides with water after color development, then soak them in 0.5% hematoxylin for 3 minutes and restain them with water until they turn blue again.

[0055] 10. Dehydration: Dehydration with different levels of alcohol (60%, 70%, 80%, 90%, 100%), 5 min per level.

[0056] 11. Transparency: Take out and place in xylene for 10 min.

[0057] 12.Seal the slides with neutral gum and observe under a microscope.

[0058] 13. Take photos of the results.

[0059] The results of the experimental group are as follows Figure 1As shown in the figure, the cell walls and nuclei of the tubular elements in the phloem at the upper part of the bamboo shoots were not stained, which is consistent with the negative control. The cell walls and / or nuclei of cells other than the phloem were brown, yellow-brown or tan. Figure 2 As shown, the nuclei of all cell types are blue, and there is no obvious staining of the cell walls.

[0060] Example 2

[0061] 1. Collection of bamboo shoots: Use common gardening shears to remove the bamboo shoots in the middle stage of development from the nodes of the underground stems, cut the bamboo shoot shells into small pieces of 0.5cm×0.5cm, and select the small piece located on the upper part of the bamboo shoot body for tissue fixation.

[0062] 2. Tissue fixation, dehydration, transparency, wax immersion, embedding, sectioning, baking, and dewaxing of sections until they are hydrated.

[0063] 3. Heat antigen retrieval: Immerse sections in EDTA antigen retrieval solution and microwave on medium heat for 8 minutes, then off heat for 5 minutes, then on low heat for 5 minutes. Cool to room temperature. After cooling, wash twice in 0.01M PBS (pH 7.2-7.6) for 5 minutes each.

[0064] 4. Add 3% H2O2 and incubate at room temperature for 10 minutes to inactivate endogenous enzymes. Rinse with PBS for 5 minutes twice.

[0065] 5. Serum blocking: Add 5% goat serum to the sections and place in a wet chamber at room temperature for 30 minutes. Wash with PBS for 5 minutes twice.

[0066] 6. Incubation with primary antibody: Add appropriately diluted primary antibody anti-H3K4me3 (A22225, rabbit polyclonal antibody, Ibotek Biotechnology Co., Ltd., Wuhan, China) (dilution ratio 1:100). Add an equal amount of PBS to the control group and incubate at 4°C overnight.

[0067] 7. Incubation with secondary antibody: Remove the slides from the refrigerator and rewarm them in a 37°C incubator for 30 min; rinse with PBS for 5 min × 2 times; add 100 μL of secondary antibody and incubate at 37°C for 30 min; rinse with PBS for 5 min × 2 times.

[0068] 8. DAB color development: Remove the slide from the incubator and rinse with PBS for 5 min × 2 times; add 100 μL of pre-prepared 0.05% color developer DAB working solution and incubate at room temperature for 1-15 min.

[0069] 9. Restaining: Wash the slides with water after color development, then soak them in 0.5% hematoxylin for 3 minutes and restain them with water until they turn blue again.

[0070] 10. Dehydration: Dehydration with different levels of alcohol (60%, 70%, 80%, 90%, 100%), 5 min per level.

[0071] 11. Transparency: Take out and place in xylene for 10 min.

[0072] 12.Seal the slides with neutral gum and observe under a microscope.

[0073] 13. Take photos of the results.

[0074] The results of the experimental group are as follows Figure 3 As shown in the figure, the cell walls and nuclei of the tubular elements of the phloem in the bamboo shoot shell, including the developing sieve elements and companion cells, showed no staining, which is consistent with the negative control. The cell walls and / or nuclei of cells other than the phloem were brown, yellow-brown or tan. The results of the control group are shown in the figure. Figure 4 As shown, the nuclei of all cell types are blue, and there is no obvious staining of the cell walls.

[0075] Example 3

[0076] 1. Collection of bamboo shoots: Use common gardening shears to remove the bamboo shoots in the later stages of development from the nodes of the underground stems, cut the bamboo shoots into small pieces of 0.5cm×0.5cm, and select the small pieces located at the top of the bamboo shoots for tissue fixation.

[0077] 2. Tissue fixation, dehydration, transparency, wax immersion, embedding, sectioning, baking, and dewaxing of sections until they are hydrated.

[0078] 3. Heat antigen retrieval: Immerse sections in EDTA antigen retrieval solution and microwave on medium heat for 8 minutes, then off heat for 5 minutes, then on low heat for 5 minutes. Cool to room temperature. After cooling, wash twice in 0.01M PBS (pH 7.2-7.6) for 5 minutes each.

[0079] 4. Add 3% H2O2 and incubate at room temperature for 10 minutes to inactivate endogenous enzymes. Rinse with PBS for 5 minutes twice.

[0080] 5. Serum blocking: Add 5% goat serum to the sections and place in a wet chamber at room temperature for 30 minutes. Wash with PBS for 5 minutes twice.

[0081] 6. Incubation with primary antibody: Add appropriately diluted primary antibody anti-H3K4me3 (A22225, rabbit polyclonal antibody, Ibotek Biotechnology Co., Ltd., Wuhan, China) (dilution ratio 1:100). Add an equal amount of PBS to the control group and incubate at 4°C overnight.

[0082] 7. Incubation with secondary antibody: Remove the slides from the refrigerator and rewarm them in a 37°C incubator for 30 min; rinse with PBS for 5 min × 2 times; add 100 μL of secondary antibody and incubate at 37°C for 30 min; rinse with PBS for 5 min × 2 times.

[0083] 8. DAB color development: Remove the slide from the incubator and rinse with PBS for 5 min × 2 times; add 100 μL of pre-prepared 0.05% color developer DAB working solution and incubate at room temperature for 1-15 min.

[0084] 9. Restaining: Wash the slides with water after color development, then soak them in 0.5% hematoxylin for 3 minutes and restain them with water until they turn blue again.

[0085] 10. Dehydration: Dehydration with different levels of alcohol (60%, 70%, 80%, 90%, 100%), 5 min per level.

[0086] 11. Transparency: Take out and place in xylene for 10 min.

[0087] 12.Seal the slides with neutral gum and observe under a microscope.

[0088] 13. Take photos of the results.

[0089] The results of the experimental group are as follows Figure 5 As shown in the figure, the cell walls and nuclei of the tubular elements of the phloem in the middle of the shoot, including sieve cells, developing sieve elements and companion cells, showed no staining, which was consistent with the negative control. The cell walls and / or nuclei of other cells except the phloem were brown, yellow-brown or tan. The results of the control group are shown in the figure. Figure 6 As shown, the nuclei of all cell types are blue, and there is no obvious staining of the cell walls.

[0090] Comparative Example 1

[0091] 1. Collection of bamboo shoots: Use common gardening shears to remove the bamboo shoots in the middle stage of development from the nodes of the underground stems, cut the bamboo shoot shells into small pieces of 0.5cm×0.5cm, and fix the tissues.

[0092] 2. Dewax the paraffin sections to water: Place the sections in xylene I for 20 minutes, xylene II for 20 minutes, anhydrous ethanol I for 10 minutes, anhydrous ethanol II for 10 minutes, 95% alcohol for 5 minutes, 90% alcohol for 5 minutes, 80% alcohol for 5 minutes, 70% alcohol for 5 minutes, and wash with distilled water.

[0093] 3. Safranin staining: Stain the sections in 1% safranin solution for 2 hours. Rinse briefly with tap water to remove excess dye.

[0094] 4. Decolorization: Decolorize in 50%, 70%, and 80% gradient alcohol for 1 minute each.

[0095] 5. Fast Green Staining: Stain the sections in 0.5% Fast Green solution for 30-60 seconds. Decolorize in anhydrous ethanol for 30 seconds and then in anhydrous ethanol for 1 minute.

[0096] 6. Sealing: Bake the sections in a 60°C oven, then transparentize in xylene for 5 minutes and seal with neutral gum.

[0097] 7. Microscopic examination, image acquisition and analysis.

[0098] The results are as follows Figure 7 As shown, the sieve elements are stained dark blue-green by Safranin Fast Green, and the shape, size, and number of the cell nuclei are unclear. The cell walls of other cells are stained light blue because they also contain cellulose, but at a lower content.

[0099] Comparative Example 2

[0100] 1. Collection of bamboo shoots: Use common gardening shears to remove the bamboo shoots in the middle stage of development from the nodes of the underground stems, cut the bamboo shoots into small pieces of 0.5cm×0.5cm, and select the small pieces located on the upper part of the bamboo shoots for tissue fixation.

[0101] 2. Tissue fixation, dehydration, transparency, wax immersion, embedding, sectioning, baking, and dewaxing of sections until they are hydrated.

[0102] 3. Heat antigen retrieval: Immerse sections in EDTA antigen retrieval solution and microwave on medium heat for 8 minutes, then off heat for 5 minutes, then on low heat for 5 minutes. Cool to room temperature. After cooling, wash twice in 0.01M PBS (pH 7.2-7.6) for 5 minutes each.

[0103] 4. Add 3% H2O2 and incubate at room temperature for 10 minutes to inactivate endogenous enzymes. Rinse with PBS for 5 minutes twice.

[0104] 5. Serum blocking: Add 5% goat serum to the sections and place in a wet chamber at room temperature for 30 minutes. Wash with PBS for 5 minutes twice.

[0105] 6. Incubate with primary antibody: add PBS dropwise and incubate in 4°C refrigerator overnight.

[0106] 7. Incubation with secondary antibody: Remove the slides from the refrigerator and rewarm them in a 37°C incubator for 30 min; rinse with PBS for 5 min × 2 times; add 100 μL of secondary antibody and incubate at 37°C for 30 min; rinse with PBS for 5 min × 2 times.

[0107] 8. DAB color development: Remove the slide from the incubator and rinse with PBS for 5 min × 2 times; add 100 μL of pre-made 0.05% color developer DAB working solution and incubate at room temperature for 15 min.

[0108] 9. Restaining: Wash the slides with water after color development, then soak them in 0.5% hematoxylin for 3 minutes and restain them with water until they turn blue again.

[0109] 10. Dehydration: Dehydration with different levels of alcohol (60%, 70%, 80%, 90%, 100%), 5 min per level.

[0110] 11. Transparency: Take out and place in xylene for 10 min.

[0111] 12.Seal the slides with neutral gum and observe under a microscope.

[0112] 13. Take photos of the results.

[0113] The results are as follows Figure 8 As shown, the cells of the phloem and xylem of the vascular tissue and the basic tissue did not react with the antibody and were not stained, and the cell nuclei appeared blue.

[0114] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A histological staining method for distinguishing phloem cells of Phyllostachys spp., characterized in that: The following steps are involved: The tissue sections of developing shoots were prepared into tissue sections. The tissue sections were used as samples and incubated with trimethylated histone antibodies, secondary antibodies, and DAB color development solution in sequence. The sections were then counterstained with hematoxylin, dehydrated with alcohol, and transparentized with xylene before being mounted for microscopic observation. The early phloem cells were not stained, while the remaining cells were stained. Prior to incubation with antibodies against trimethylated histones, antigen retrieval, inactivation of endogenous enzymes, and serum blocking were also performed.

2. The histological staining method according to claim 1, characterized in that: The bamboo shoot bud tissue comprises the following steps: removing 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 fixed, dehydrated, transparent, soaked in wax, embedded, sliced, baked, and dewaxed to water to prepare tissue sections.

4. The histological staining method according to claim 1, 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 adding 5% by volume sheep serum to the tissue slices and then leaving the slices to stand at room temperature.

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

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

7. The histological staining method according to claim 6, characterized in that: The dilution ratio of the anti-H3K4me3 is 1:50~200.

8. Use of the histological staining method according to any one of claims 1 to 7 in distinguishing phloem cells in plants of the genus Phyllostachys.

Images