CN110455595B - Plant tissue integral staining method for ultrathin section - Google Patents

Plant tissue integral staining method for ultrathin section Download PDF

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CN110455595B
CN110455595B CN201910644850.8A CN201910644850A CN110455595B CN 110455595 B CN110455595 B CN 110455595B CN 201910644850 A CN201910644850 A CN 201910644850A CN 110455595 B CN110455595 B CN 110455595B
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ethanol
tch
acetone
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acetate
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CN110455595A (en
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李晓娟
姚小敏
林金星
马玲玉
沈微微
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Beijing Forestry University
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/36—Embedding or analogous mounting of samples
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/36—Embedding or analogous mounting of samples
    • G01N2001/364—Embedding or analogous mounting of samples using resins, epoxy

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Abstract

本发明公开一种用于超薄切片的植物组织整体染色法。该方法包括如下实验步骤：(1)固定：收集成熟的青杄花粉，用戊二醛与多聚甲醛进行前固定，再使用1％锇酸进行后固定；用TCH处理，再用1％锇酸进一步沉积锇；(2)块染：用2.5％醋酸钆、2.5％醋酸钐、2％醋酸双氧铀进行块染；(3)乙醇梯度脱水：分别用30％、50％、60％、70％、80％、90％、95％、100％乙醇进行脱水；(4)渗透：分别用乙醇与丙酮混合溶液、100％丙酮、丙酮与spurr树脂混合溶液、100％spurr树脂进行渗透处理；(5)包埋与聚合：在包埋板中加入100％spurr树脂，放入样品后置于烘箱进行聚合。本方法具操作简单、染色效果较好，图像对比度明显；还有利于超薄连续切片，为实现植物组织三维重构奠定了技术基础。

Figure 201910644850

The invention discloses a whole plant tissue dyeing method for ultra-thin sectioning. The method includes the following experimental steps: (1) Fixation: collecting mature pollen of A. chinensis, pre-fixation with glutaraldehyde and paraformaldehyde, and post-fixation with 1% osmic acid; treatment with TCH, and then with 1% osmium (2) block staining: block staining with 2.5% gadolinium acetate, 2.5% samarium acetate, 2% uranyl acetate; (3) ethanol gradient dehydration: use 30%, 50%, 60%, 70%, 80%, 90%, 95%, 100% ethanol for dehydration; (4) Permeation: infiltrate with ethanol and acetone mixed solution, 100% acetone, acetone and spurr resin mixed solution, and 100% spurr resin respectively; (5) Embedding and polymerization: 100% spurr resin was added to the embedding plate, and the sample was placed in an oven for polymerization. The method has the advantages of simple operation, good staining effect, and obvious image contrast; it is also beneficial to ultra-thin serial sectioning, and lays a technical foundation for realizing three-dimensional reconstruction of plant tissue.

Figure 201910644850

Description

Plant tissue integral staining method for ultrathin section
Technical Field
The invention relates to a plant tissue integral staining method for ultrathin sections
Background
Ultrathin sectioning techniques are important methods for studying cellular sub-microstructures. However, in order to obtain a sharp image, the biological sample must be electronically stained. The electronic staining is to bind or adsorb heavy metals in heavy metal salts such as uranium, lead, osmium, and tungsten to some components in the tissue to improve the contrast of the microstructure in the sample. Heavy metal atoms with different numbers are adsorbed on different structural components, areas with more bound heavy metals (namely, areas with compact structure and high atomic number) have strong electron scattering capacity, and are in black with compact electron microscope under the electron microscope, areas with less bound heavy metals are in light black and gray black, and areas without bound heavy metals are in electron transparent areas.
The current common dyeing method is slice dyeing, namely fishing ultrathin slices on a copper net for electronic dyeing. However, sheet dyeing has a number of limitations, such as: in the large-batch net-carrying dyeing process, the net-carrying quantity of each dyeing is limited, and even if the nets are carried in the same batch, the dyeing time is not easy to be uniform; the operation is complicated, the grid is easy to bend and deform, and the focusing effect during the observation of an electron microscope is influenced; due to the fact that liquid is replaced for multiple times, ultrathin slices on the carrying net are prone to damage, and phenomena such as folding and slice dropping occur; in the dyeing process, the lead dye solution is easily combined with carbon dioxide in the air to generate pollution, so that the observation effect is influenced; the method can not carry out large-scale continuous film collection on the specimen, and is an important barrier for realizing three-dimensional reconstruction. Aiming at the phenomenon, an integral dyeing method can be adopted in the preparation process of the electron microscope sample, namely, the electron dyeing is carried out in the sample preparation process, and the secondary dyeing is not needed after the section.
In the past, studies on bulk staining have mostly used animal tissues as materials. However, plant tissues, unlike animal tissues, have cell walls, which is a feature that causes great trouble in the overall staining of plant tissues. Therefore, it is of great significance to study the whole plant tissue staining method for ultrathin sections.
Disclosure of Invention
The invention aims to increase lipid staining through a Thiocarbohydrazide (TCH) bridging agent, and the method does not need staining after slicing, is convenient to operate, and is beneficial to realizing continuous slicing of plant tissues and three-dimensional reconstruction.
The invention provides a plant tissue integral staining method for ultrathin sections. The method uses picea wilsonii pollen as a material. The method specifically comprises the following steps:
(1) fixing the plant material; placing mature picea wilsonii pollen in a fixing solution prepared from glutaraldehyde and paraformaldehyde for pre-fixing, washing with a PBS buffer solution, and then performing post-fixing with 1% osmic acid;
(2) osmium deposition is carried out by TCH; treating with 1% TCH, standing at room temperature for 30min-1h, soaking with 1% osmic acid, and further depositing osmium;
(3) block dyeing: respectively carrying out block dyeing on the plant sample by using 2.5% gadolinium acetate, 2.5% samarium acetate and 2% uranyl acetate, and standing for 8-12 hours at 4 ℃ or standing for 4 hours at normal temperature;
(4) and (3) dehydrating: performing ethanol gradient dehydration on the block-dyed plant sample, and performing gradient dehydration by using 30%, 50%, 60%, 70%, 80%, 90%, 95% and 100% ethanol respectively;
(5) and (3) infiltration: respectively carrying out permeation treatment by using a mixed solution of ethanol and acetone, 100% acetone, a mixed solution of acetone and spurr resin and 100% spurr resin;
(6) embedding and polymerization: adding 100% spurr resin into the embedding plate, putting the plant sample, adjusting the position, and putting the plant sample in an oven for polymerization.
Preferably, in step (1): the concentration of the PBS buffer solution is 0.1M, and the pH value is 7.2; the fixing solution is a mixed solution of 2.4% paraformaldehyde and 2% glutaraldehyde; the PBS buffer solution and the stationary solution are stored in a refrigerator at 4 ℃ and are placed on ice when in use; after the pre-fixation, the plant sample after the pre-fixation is washed for 3 times by PBS buffer solution, each time is 10-15min, and the fixing solution is fully washed away; post-fixation was performed with 1% osmic acid, left at 4 ℃ for 2h, after which it was necessary to wash 3 times with purified water for 10-15min each.
Preferably, in step (2): weighing 0.05g of TCH, dissolving the TCH in 5mL of purified water, wrapping a layer of tinfoil to prevent light, placing the prepared 1% TCH in an oven at 60 ℃ for 1h, shaking once every ten minutes, and finally filtering the TCH by using a filter with the pore diameter of 0.22 mu m for use; adding 300 μ l TCH, standing at room temperature for 30min-1h to avoid TCH precipitation at low temperature, cleaning plant sample treated with TCH with purified water for 3 times, each time for 10-15min, performing osmium deposition with 1% osmate, standing at room temperature for 1h, and cleaning with purified water for 3 times, each time for 10-15 min.
Preferably, in step (3): the block dye of 2.5 percent gadolinium acetate, 2.5 percent samarium acetate and 2 percent uranyl acetate are all prepared by purified water, and the concentration is mass percent.
Preferably, in step (4): performing gradient dehydration with 30%, 50%, 60%, 70%, 80%, 90%, 95%, and 100% ethanol respectively, and dehydrating with 100% ethanol for 3 times for 15min to fully dehydrate the sample, wherein the ethanol content is 30%, 50%, 60%, 70%, 80%, 90%, and 95%, and the solvent is purified water.
Preferably, in step (5): respectively treating with solutions of ethanol and acetone at volume ratios of 2:1, 1:1 and 1:2 for 15min, then treating with 100% acetone for 3 times, each time for 15min, respectively treating with solutions of acetone and spurr resin at volume ratios of 2:1, 1:1 and 1:2 for 3h, then treating with 100% spurr resin for at least 3 times, and changing the liquid every 12 h.
Preferably, the recipe of the spurr resin is 26g of NSA hardener (nonylsuccinic anhydride), 10g of ERL4221 resin (vinylcyclohexene dioxide), 6g of DER 736 plasticizer (diethylene glycol) and 0.4g of DMAE catalyst (methylene glycol); in the step (6): the polymerization was carried out for 2h at 40 ℃ and then for 8h at 70 ℃.
The feature of the present invention is the use of TCH as a bridging agent during osmium impregnation, which attaches itself to the osmium already present after initial osmium fixation and acts as a bridge, allowing more osmium to be deposited at the original osmium sites. Therefore osmium-tropic TCH enhances osmium staining of lipid components. And then, a block dyeing agent is used for dyeing, so that the electronic contrast of the internal structure of the cell is effectively enhanced, the problems of lead pollution caused by lead citrate block dyeing and a great deal of inconvenience caused by slice dyeing are solved, and technical support is provided for ultrathin continuous slicing of plant tissues.
Drawings
FIG. 1 is picea wilsonii pollen dyed with 2.5% gadolinium acetate blocks, and FIG. 2 is a partial enlarged view thereof; FIG. 3 is a picea wilsonii pollen stained with 2.5% samarium acetate blocks, and FIG. 4 is a partial enlarged view thereof; fig. 5 is picea wilsonii pollen dyed with 2% uranyl acetate block, and fig. 6 is a partial enlarged view thereof.
Detailed Description
Sample collection
Collecting mature picea wilsonii pollen, and standing at-20 deg.C for use.
Sample preparation
1. Front fixing: and (3) placing the collected mature picea wilsonii pollen in a fixing solution consisting of 2% of glutaraldehyde and 2.4% of paraformaldehyde for fixing, and vacuumizing for 1-2 h. Note that the fixative was stored in a 4 ℃ freezer and placed on ice when used.
2. Rinsing: rinsing the pre-fixed plant sample with 0.1M phosphate buffer solution, and washing for 3 times, each time for 10-15 min; the phosphate buffer was stored in a refrigerator at 4 ℃ and placed on ice when used.
3. Post-fixing: the rinsed plant samples were post-fixed with 1% osmic acid and placed at 4 ℃ for 2 h.
4. Rinsing: washing the fixed plant sample with purified water for 3 times, each for 10-15 min.
5. Preparing 1% TCH: weighing 0.05g TCH, dissolving in 5mL of purified water, wrapping a layer of tinfoil to prevent light, placing the prepared 1% TCH in an oven at 60 ℃ for 1h, shaking once every ten minutes, and finally filtering with a filter with the pore diameter of 0.22 mu m for later use.
6. TCH treatment: and (4) treating the rinsed plant sample by using 1% TCH, and standing for 30min-1h at room temperature.
7. Rinsing: and washing the plant sample treated by TCH with purified water for 3 times, 10-15min each time.
8. Deposition of osmium: the rinsed plant samples were soaked with 1% osmic acid and left at room temperature for 1 h.
9. Rinsing: and (3) washing the plant sample subjected to osmium deposition with purified water for 10-15min each time.
10. Block dyeing: and (3) respectively carrying out block dyeing on the rinsed plant sample by using 2.5% gadolinium acetate, 2.5% samarium acetate and 2% uranyl acetate, and standing at 4 ℃ for 8-12h or at room temperature for 4 h.
11. Rinsing: washing the block-dyed plant sample with purified water for 3 times, each for 10-15 min.
12. And (3) dehydrating: the rinsed plant samples were dehydrated with 30%, 50%, 60%, 70%, 80%, 90%, 95%, 100% ethanol for 15min each time in a gradient manner, and 3 times with 100% ethanol.
13. Ethanol and acetone infiltration: treating with solutions of ethanol and acetone at volume ratio of 2:1, 1:1, and 1:2 for 15min, respectively, and treating with 100% acetone for 15min for 3 times.
14. Acetone penetration with spurr resin: treating with acetone and spurr resin at volume ratio of 2:1, 1:1, and 1:2 for 3 hr, respectively, and treating with 100% spurr resin for at least 3 times, and changing liquid every 12 hr.
15. Embedding and polymerization: firstly, 100% spurr resin is added into an embedding plate, then a plant sample is placed, the position of the plant sample is adjusted, and the plant sample is placed in an oven. Polymerization was first carried out at 40 ℃ for 2h and then at 70 ℃ for 8 h.
And adding a silica gel drying agent into the prepared sample, placing the sample into a sealed bag, and storing the sample at room temperature.
It will be understood by those skilled in the art that the foregoing is only a preferred embodiment of the present invention, and is not intended to limit the invention, and that any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the present invention.

Claims (6)

1. A plant tissue bulk staining method for ultrathin sections comprises the following steps:
(1) fixing the plant material; placing mature picea wilsonii pollen in a fixing solution prepared from 2% glutaraldehyde and 2.4% paraformaldehyde for pre-fixing, washing the pre-fixed plant sample with PBS buffer solution for 3 times, 10-15min each time, sufficiently washing off the fixing solution, washing with PBS buffer solution with concentration of 0.1M and pH of 7.2, post-fixing with 1% osmic acid, standing at 4 deg.C for 2h, and washing with purified water for 3 times, 10-15min each time;
(2) osmium deposition is carried out by TCH; treating with 1% by mass of TCH, standing at room temperature for 30min-1h, soaking with 1% osmic acid, and further depositing osmium;
(3) block dyeing: respectively carrying out block dyeing on the plant sample by using 2.5% gadolinium acetate, 2.5% samarium acetate and 2% uranyl acetate, and standing for 8-12 hours at 4 ℃ or standing for 4 hours at normal temperature; wherein, the block dyes of 2.5 percent of gadolinium acetate, 2.5 percent of samarium acetate and 2 percent of uranyl acetate are all prepared by purified water, and the concentration is mass percent;
(4) and (3) dehydrating: performing ethanol gradient dehydration on the block-dyed plant sample, and performing gradient dehydration by using 30%, 50%, 60%, 70%, 80%, 90%, 95% and 100% ethanol respectively; wherein 30%, 50%, 60%, 70%, 80%, 90% and 95% ethanol are in volume concentration, and the solvent is purified water;
(5) and (3) infiltration: respectively carrying out permeation treatment by using a mixed solution of ethanol and acetone, 100% acetone, a mixed solution of acetone and spurr resin and 100% spurr resin;
(6) embedding and polymerization: adding 100% spurr resin into the embedding plate, putting the plant sample, adjusting the position, and putting the plant sample in an oven for polymerization.
2. The bulk dyeing method according to claim 1, characterized in that in step (1): the PBS buffer solution and the fixing solution are both required to be stored in a refrigerator at 4 ℃ and are placed on ice when in use.
3. The bulk dyeing method according to claim 1, characterized in that in step (2): weighing 0.05g TCH, dissolving in 5mL of purified water, wrapping a layer of tinfoil to prevent light, placing the prepared 1% TCH in a 60 ℃ oven for 1h, shaking once every ten minutes, and finally filtering with a filter with the pore diameter of 0.22 mu m for use; adding 300 μ l TCH, standing at room temperature for 30min-1h to avoid TCH precipitation at low temperature, cleaning plant sample treated with TCH with purified water for 3 times, each time for 10-15min, performing osmium deposition with 1% osmate, standing at room temperature for 1h, and cleaning with purified water for 3 times, each time for 10-15 min.
4. The bulk dyeing method according to claim 1, characterized in that in step (4): gradient dehydration with 30%, 50%, 60%, 70%, 80%, 90%, 95%, 100% ethanol, respectively, for 15min, and 100% ethanol for 3 times, respectively, to fully dehydrate the sample.
5. The bulk dyeing method according to claim 1, characterized in that in step (5): treating with solutions of ethanol and acetone at volume ratios of 2:1, 1:1, and 1:2 for 15min, respectively, treating with 100% acetone for 3 times, 15min each time, treating with solutions of acetone and spurr resin at volume ratios of 2:1, 1:1, and 1:2 for 3h, respectively, treating with 100% spurr resin for at least 3 times, and changing the liquid every 12 h.
6. The bulk dyeing process according to claim 1, characterized in that the spurr resin formulation is NSA hardener 26g, ERL4221 resin 10g, DER 736 plasticizer 6g, DMAE catalyst 0.4 g; in the step (6): the polymerization was carried out for 2h at 40 ℃ and then for 8h at 70 ℃.
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CN112665951B (en) * 2020-12-22 2022-11-11 中国农业科学院作物科学研究所 Embryo milk tissue embedding method and application
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