A method for preparing paraffin sections of highly lignified and highly heterogeneous materials

Abstract

The present invention discloses a method for preparing paraffin sections of highly lignified and highly heterogeneous materials, relating to the technical field of sectioning. The method of the present invention can be widely used to prepare paraffin sections of highly lignified and highly heterogeneous materials of different ages and species. This method overcomes the problems of cracked, curled, and incomplete sections currently encountered when preparing highly lignified and highly heterogeneous materials using paraffin sections. The sections produced by the present invention have a complete and clear structure and are highly applicable, laying the foundation for observing the secondary growth process of tree stems.

Description

Technical Field

[0001] The invention relates to the technical field of slicing, in particular to a method for preparing paraffin slices of highly lignified and highly heterogeneous materials. Background Art

[0002] Currently, paraffin sectioning studies of tree vascular bundles typically focus on young saplings or young branches to observe the differentiation of primary structures. Studies of secondary vascular bundles in mature trees typically use micro-growth cones to drill micro-cores for paraffin sectioning. For example, Zhang Junzhou's micro-core paraffin sectioning technique involves drilling micro-cores with micro-growth cones, fixing them with FAA, softening them with the glycerol-ethanol method, dehydrating them with conventional ethanol, clearing them with xylene, then embedding them in wax, sectioning them, and staining and mounting them. Overall, this method remains largely unchanged from conventional methods.

[0003] The most effective method for preparing hard paraffin sections is Zhou Naifu's method for preparing paraffin sections of non-homogeneous woody plant tissue. This method uses tert-butyl alcohol instead of ethanol and xylene, simplifying the paraffin sectioning process and proposing a softening method using a combination of hydrogen peroxide-glacial acetic acid and tert-butyl alcohol-glycerol. The steps of this method are as follows:

[0004] 1. Fix the material with a modified fixative (70% tert-butyl alcohol: 35%–40% formaldehyde: propionic acid: glycerol = 85:5:5:5, v / v / v / v) and evacuate until the material sinks. Fix for at least 48 hours.

[0005] 2. Place the stem segments in a hydrogen peroxide-glacial acetic acid mixed solution (1:1, v / v), vacuum for 30 minutes, and treat for 3 hours. Then transfer the material to a covered glass bottle filled with a 70% tert-butyl alcohol-propylene glycol mixed solution (1:1, v / v) and place it in a 50°C constant temperature incubator for 2 days.

[0006] 3. Use the tert-butanol gradient dehydration method, i.e., 50% tert-butanol for 1.5 hours → 70% tert-butanol for 1.5 hours → 85% tert-butanol for 1.5 hours → 95% tert-butanol for 1 hour → pure tert-butanol for 30 minutes → pure tert-butanol for 30 minutes. Vacuum treatment is performed after changing the dehydrating agent.

[0007] 4. Immerse the stem segments in a 50% paraffin solution (tert-butyl alcohol: paraffin = 1:1, v / v) for 6 h at 54–56°C. Then, transfer to a 75% paraffin solution (tert-butyl alcohol: paraffin = 1:3, v / v) for 6 h at 56–58°C. Finally, transfer to pre-melted pure paraffin at 58–60°C. Change the paraffin solution every 4 h for three times. This entire process should be performed in a constant temperature chamber. Sections should then be routinely sectioned, stained, and mounted.

[0008] This method has been shown to be effective for producing paraffin sections of low-lignified materials. However, it still has significant limitations. Tert-butyl alcohol is less transparent than xylene, and the softening method used is relatively gentle. When the experimental material is highly lignified, the wax strips are prone to breakage, and the sections produced are often incomplete. Summary of the Invention

[0009] The purpose of the present invention is to provide a method for preparing paraffin slices of highly lignified and highly heterogeneous materials, so as to solve the problems existing in the above-mentioned prior art and realize the preparation of paraffin slices of highly lignified and highly heterogeneous materials.

[0010] To achieve the above object, the present invention provides the following solutions:

[0011] The present invention provides a method for preparing paraffin sections of highly lignified and highly heterogeneous materials, comprising the following steps:

[0012] 1) Sampling and fixation

[0013] After sampling, the material was placed in FAA fixative, vacuumed, and then placed in a 4°C refrigerator for more than 72 hours;

[0014] 2) Softening

[0015] The fixed material is placed in a hydrochloric acid-ethanol softening solution or a NaOH-ethanol softening solution for a first softening, and then the softening solution is replaced with 70 vol% ethanol. After the replacement is completed, the material is placed in a glycerol-ethanol softening solution for a second softening; the hydrochloric acid-ethanol softening solution is a mixture of 36% concentrated hydrochloric acid and anhydrous ethanol in a volume ratio of 2:3;

[0016] The NaOH-ethanol softening solution is a mixture of NaOH and 70 vol% ethanol in a mass volume ratio of 24 g:100 mL;

[0017] The glycerol-ethanol softening liquid is a mixture of 70 vol% ethanol and glycerol in a volume ratio of 1:1;

[0018] Preferably, the first softening treatment time is 2 hours, and the second softening treatment time is 2 days;

[0019] 3) Dehydration

[0020] After the material is softened, it is dehydrated using ethanol solution;

[0021] Specifically: the material was placed in 70 vol% ethanol for 3 times, each time for 2 hours. After the last treatment, the material was soaked in 70 vol% ethanol at room temperature overnight; the next day, the material was dehydrated step by step with an ethanol concentration and time gradient of 70%-2 hours, 85%-2 hours, 95%-2 hours, 100%-1 hour, and 100%-1 hour.

[0022] 4) Transparency

[0023] The dehydrated material was transitioned in a 50 vol% xylene + 50 vol% anhydrous ethanol solution, then transparentized with xylene, and then placed in a 50 vol% xylene + 50 vol% paraffin mixed solution at 40°C overnight;

[0024] 5) Wax dipping

[0025] The material was wax-impregnated at 60°C;

[0026] 6) Embedding

[0027] Embed the material after wax immersion in paraffin wax;

[0028] 7) Slicing, patching and baking

[0029] The embedded material was placed in a 50 vol% glycerol + 50 vol% aqueous solution, and then sliced, mounted, baked, and then stained;

[0030] After the baking is completed, the material is dewaxed with xylene and transitioned with 50 vol% xylene + 50 vol% anhydrous ethanol, then rehydrated, dyed with safranin solution, and then dehydrated and dyed with fast green.

[0031] The rehydration was performed in the order of two doses of anhydrous ethanol for 3 min, 95 vol% ethanol for 3 min, 85 vol% ethanol for 3 min, 70 vol% ethanol for 3 min, and 50 vol% ethanol for 3 min.

[0032] After the safranin staining was completed, the cells were dehydrated in stages using 50 vol% ethanol, 70 vol% ethanol, 85 vol% ethanol, and 95 vol% ethanol for 2 min each time.

[0033] 8) Sealing

[0034] Furthermore, the temperature of the first softening is 55° C. and the time is 1-4 hours.

[0035] Furthermore, the first softening time is 2 hours.

[0036] Furthermore, the temperature of the second softening is 50° C. and the time is 2-5 days.

[0037] Furthermore, the second softening time is 2 days.

[0038] Furthermore, step 8) the specific steps of the sealing treatment are: the stained sections are sequentially treated with 95 vol% ethanol solution, 100 vol% ethanol solution, 50 vol% xylene + 50 vol% anhydrous ethanol, and 2 times of xylene solution, and then sealed with neutral gum.

[0039] The processing time for each step in the step-by-step processing is 2 minutes.

[0040] Currently, paraffin sectioning is commonly used to observe tree vascular bundles using sections of young saplings or young branches. While the paraffin production process for these materials is relatively mature, this method is only suitable for exploring primary structural changes and tissue differentiation processes at a young age. The vascular tissue structure, tissue differentiation, and cellular lignification deposition characteristics of older trees differ significantly from those of younger saplings and young branches. Currently, paraffin sectioning using micro-cores is a common method for observing secondary vascular bundles in large trees, but this method still has significant limitations. Paraffin sectioning remains difficult when using large blocks, hardy species, or irregular, highly lignified, and heterogeneous materials grown in the wild. Consequently, various embedding agents and microtome combinations have been developed to produce rigid sections. However, producing high-quality, highly lignified sections using paraffin sectioning remains a difficult problem.

[0041] While the method disclosed by Zhou Naifu et al. can produce materials with a low degree of lignification, it is less effective when producing highly lignified and highly heterogeneous materials, often resulting in incomplete and damaged sections, making it difficult to obtain high-quality paraffin sections. To address this problem, the present invention provides a method for producing paraffin sections that can be widely used for producing highly lignified and highly heterogeneous plant materials of different ages and species. This method overcomes the problems of cracking, curling, and incomplete sections currently encountered when producing such highly lignified materials using paraffin sections. Experiments have confirmed that the resulting sections have a complete and clear structure and are highly applicable, laying the foundation for observing the secondary growth process of tree stems.

[0042] The present invention discloses the following technical effects:

[0043] The present invention provides a paraffin section production method that can be widely used for highly lignified and highly heterogeneous materials of different age classes and tree species, thereby softening the material while ensuring the integrity of the fragile tissue of the section to obtain high-quality hard sections. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0045] Figure 1 Paraffin sections of the xylem and bark of a Masson pine tree over 30 years old and with a diameter at breast height of 30 cm or more, prepared in Example 1 of the present invention, wherein (a) is the xylem and (b) is the bark;

[0046] Figure 2 Paraffin sections of vascular bundles of cedarwood with a diameter at breast height of 30 cm+ prepared in Example 2 of the present invention;

[0047] Figure 3 Paraffin sections of cedar vascular bundles with a diameter at breast height of 50 cm+ prepared in Example 2 of the present invention;

[0048] Figure 4 Paraffin sections of the stems of one-year-old Masson pine seedlings prepared in Example 3 of the present invention;

[0049] Figure 5 The paraffin sections prepared in Comparative Example 1 of the present invention;

[0050] Figure 6 This is a slice sample prepared in Comparative Example 2 of the present invention;

[0051] Figure 7 The slices of comparative example 3 of the present invention were not made with a wooden block base and were softened overtime;

[0052] Figure 8 The slices are prepared in Comparative Example 4 of the present invention but not soaked in 50% glycerol. DETAILED DESCRIPTION

[0053] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0054] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0055] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0056] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0057] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0058] The slice preparation method of the present invention comprises the following steps:

[0059] 1) Sampling and fixation

[0060] After sampling, place in FAA fixative, evacuate and place in 4℃ refrigerator for more than 72h;

[0061] 2) Softening

[0062] In order to adapt to materials of different hardness, the softening process of the present invention provides two softening liquid combinations (strong softening liquid + weak softening liquid):

[0063] ① Softening solution combination for cell structure observation:

[0064] (Hydrochloric acid-ethanol) + (glycerol-ethanol) = (36% concentrated hydrochloric acid: anhydrous ethanol, volume ratio = 2:3) + (70% ethanol: glycerol, volume ratio = 1:1). Advantages: Softens by breaking down lignin, significantly reducing slice hardness. Suitable for materials of varying hardness, maintains intact tissue structure, and achieves excellent slice results. Disadvantages: Reacts with cellular sugars and is not recommended if preserving sugars is a concern.

[0065] ② Softening solution combination to retain cell contents:

[0066] (NaOH-ethanol) + (glycerol-ethanol) = (NaOH to 70 vol% ethanol, mass / volume ratio = 24 g:100 mL) + (70 vol% ethanol:glycerol, volume ratio = 1:1). Advantages: Suitable for most hard materials, good softening effect. Minimal damage to cellular contents and structural carbohydrates, retaining polysaccharides within cells. Disadvantages: Slightly lower softening ability than combination ①.

[0067] Specifically, the NaOH-ethanol softening solution can be obtained by dissolving 24 g of NaOH in 100 mL of 70 vol% ethanol.

[0068] The softening steps are as follows:

[0069] Remove the material from the fixing solution and place it in a glass bottle. Pour in hydrochloric acid-ethanol softening solution or NaOH-ethanol softening solution. The softening solution should just cover the material, preferably not more than half the volume of the bottle. Place it in a 55℃ oven for 2 hours (it can be adjusted appropriately according to the hardness of the material. After exploring the softening treatment time, 1 hour is sufficient for low-hardness materials. NaOH-ethanol softening should not exceed 4 hours, and hydrochloric acid-ethanol should not exceed 3 hours). After cooling to room temperature, place it in 70vol% ethanol for 2 hours to replace the softening solution. After replacing it twice in an oven at 35℃, let the material stand in 70vol% ethanol at room temperature overnight. After the softening solution is completely replaced, place the material in glycerol-ethanol softening solution. The softening solution should not exceed half the volume of the glass bottle. After sealing, place it in a 50℃ oven for 2 days (it can be extended appropriately according to the hardness, preferably not more than 5 days).

[0070] 3) Dehydration

[0071] After the softening of the material was completed, it was placed in 70 vol% ethanol for dehydration and softening liquid replacement three times, each time for 2 hours. The first two times were placed in a 35°C oven for treatment, and the last time was placed at room temperature overnight. The next day, dehydration was carried out step by step with 70%-2h, 85%-2h, 95%-2h, 100%-1h, and 100%-1h ethanol concentration (volume concentration) and time gradient;

[0072] 4) Transparency

[0073] After dehydration, the material was transitioned in a 50 vol% xylene + 50 vol% anhydrous ethanol solution for 2 h. After the transition was completed, it was transparentized twice in a pure xylene solution, each time for 2 h. After the transparentization was completed, it was placed in a mixed solution of 50 vol% xylene + 50 vol% paraffin = 1:1 and stored in a constant temperature box at 40°C overnight;

[0074] 5) Wax dipping

[0075] The whole process of wax dipping was treated with pure wax (liquid paraffin after heating and dissolving) at a temperature of 60°C. The pure wax was replaced three times at intervals of 4h, 2h, and 2h respectively. Finally, the wax was placed in a 60°C oven and immersed in paraffin overnight.

[0076] 6) Embedding

[0077] Embed the material the next day after the wax soaking is complete. Pour the melted paraffin into the embedding box for embedding. Heat the tweezers with an alcohol lamp and use the tweezers to stir the material in the embedding box to remove bubbles. Then put the material into the ice box and adjust the position of the material as needed. Cool it at room temperature. After the wax block is completely solidified, take out the sample and make sure it is parallel to each other from front to back and left to right. Let the melted wax adhere to the wood block. It should adhere tightly and not fall off.

[0078] The embedding base is made of wood. Conventional embedding cassettes lack hardness and density, and the wax block attached to the base softens when in a water bath. This can cause slight deformation during sectioning due to the hard material, affecting the sectioning effect. The wax block is then directly bonded with high-temperature melted paraffin. After connection, high-temperature paraffin is titrated around the wax block to reinforce it, ensuring that the wax block does not fall off during sectioning.

[0079] 7) Slicing, patching and baking

[0080] Before slicing, prepare a 50% glycerol solution in a 1:1 volume ratio of glycerol to distilled water. Slice the sample on a microtome. After cutting to the desired section, invert the sample and place it in the 50% glycerol solution. Let it rest at room temperature for at least 12 hours, but it can be left there for longer periods of time (multiple days, the longer the better). After this resting period, begin sectioning. If the material is extremely hard and the results are still poor, place the wax block inverted in the 50% glycerol solution and heat it at 37-40°C for at least 10 minutes to further soften the material and reduce friction between the material and the blade. Use a microtome to cut the wax block into 8-12 μm wax strips. Drop the adhesive film onto a glass slide and mix evenly. Gently place the wax strip on the glass slide and spread the slide on a slide spreader at 48°C. After the wax strip is fully spread, tilt the slide and absorb the excess adhesive film with absorbent paper. Then, dry the slide in a 40°C oven for 1-2 days before staining.

[0081] Specifically, dyeing can be performed in the following ways:

[0082] Sections were dewaxed twice in pure xylene for 10 minutes each, then transitioned in 50 vol% xylene + 50 vol% anhydrous ethanol for 3 minutes. Sections were then rehydrated in anhydrous ethanol (3 minutes twice), 95% ethanol (3 minutes), 85% ethanol (3 minutes), 70% ethanol (3 minutes), and 50% ethanol (3 minutes) in this order. Sections were then stained with safranin for 14 hours. Following safranin staining, sections were dehydrated in 50 vol% ethanol, 70 vol% ethanol, 85 vol% ethanol, and 95 vol% ethanol for 2 minutes each. Sections were then stained with Fast Green for 15-20 seconds.

[0083] 8) Seal the slide

[0084] The stained sections were sequentially treated with 95 vol% ethanol solution, 100 vol% ethanol solution, 50 vol% xylene + 50 vol% anhydrous ethanol, and two times of pure xylene, each for 2 min, and then sealed with neutral gum.

[0085] The present invention will be further described in detail below with reference to the embodiments:

[0086] Example 1

[0087] Preparation of paraffin sections of the xylem and bark of Pinus massoniana trees over 30 years old and with a diameter at breast height of 30 cm or more (the raw material is characterized by a high degree of lignification and heterogeneity, and contains a large number of stone cells):

[0088] 1) Sampling and fixation

[0089] The xylem and bark layers were sampled separately and placed in FAA fixative, vacuumed, and then placed in a 4°C refrigerator for 72 hours.

[0090] 2) Softening

[0091] The softening treatment was carried out using a combination of NaOH-ethanol + glycerol-ethanol softening liquid, wherein the NaOH-ethanol softening liquid was prepared by dissolving 24g NaOH in 100ml 70vol% ethanol; the glycerol-ethanol softening liquid was a mixture of 70vol% ethanol and glycerol in a volume ratio of 1:1.

[0092] The softening steps are as follows:

[0093] Remove the material from the fixative and place it in a glass bottle. Pour in a NaOH-ethanol softening solution, just enough to submerge the material and not exceeding half the bottle's volume. Place the sample in a 55°C oven for 3 hours. After the softening solution treatment is completed, let it stand at room temperature. After the temperature drops to room temperature, pour out the softening solution, pour in 70 vol% ethanol, and place it in a 35°C oven to replace the remaining softening solution in the sample. This is done every 2 hours. After two replacements, the sample is placed in a 35°C oven overnight. The next day, the 70 vol% ethanol is poured out and replaced with a glycerol-ethanol softening solution in a 50°C oven for 3 days. The sample is then placed in 70 vol% ethanol and replaced in a 35°C oven to replace the remaining softening solution. This is repeated three times, each for 2 hours, and the third time is left at room temperature overnight.

[0094] 3) Dehydration

[0095] The next day, dehydration was performed step by step with ethanol concentration (volume concentration) and time gradient of 70%-2h, 85%-2h, 95%-2h, 100%-1h, and 100%-1h;

[0096] 4) Transparency

[0097] After dehydration, the material was transitioned in a 50 vol% xylene + 50 vol% anhydrous ethanol solution for 2 h. After the transition was completed, it was transparentized twice in a pure xylene solution, each time for 2 h. After the transparentization was completed, it was placed in a mixed solution of 50 vol% xylene + 50 vol% paraffin and stored in a constant temperature box at 40°C overnight.

[0098] 5) Wax dipping

[0099] Pure wax was used throughout the wax dipping process at a temperature of 60°C. Pure wax was replaced three times with intervals of 4 h, 2 h, and 2 h, respectively. Finally, the paraffin was immersed overnight.

[0100] 6) Embedding

[0101] Embed the material the next day after the wax soaking is complete. Pour the melted paraffin into the embedding box for embedding. Heat the tweezers with an alcohol lamp and use the tweezers to stir the material in the embedding box to remove bubbles. Then put the material into the ice box and adjust the position of the material as needed. Cool it at room temperature. After the wax block is completely solidified, take out the sample and make sure it is parallel to each other from front to back and left to right. Let the melted wax adhere to the wood block. It should adhere tightly and not fall off.

[0102] 7) Slicing, patching and baking

[0103] Before sectioning, slice the sample on a microtome using a 50 vol% glycerol + 50 vol% distilled water solution. Once the desired section is reached, invert the sample and place it in a 50 vol% glycerol + 50 vol% distilled water solution to soak overnight at room temperature. Use a microtome to cut the wax block into 10 μm strips. Drop the adhesive film onto a glass slide and mix thoroughly. Gently place the strip on the glass slide and spread it on a slide spreader at 48°C. Once the strip is fully spread, tilt the slide and remove any excess adhesive film with absorbent paper. Dry the slide in a 40°C oven for 24 hours before staining with phloroglucinol.

[0104] 8) Seal the slide

[0105] The stained sections were sequentially treated with 95 vol% ethanol solution, 100 vol% ethanol solution, 50 vol% xylene + 50 vol% anhydrous ethanol solution, two times of pure xylene, and pure xylene for 2 min each time, and then sealed with neutral gum.

[0106] Figure 1 Paraffin sections of the xylem and fallen bark of a Masson pine tree with a tree age of more than 30 years and a diameter at breast height of 30 cm+ were prepared, wherein (a) is the xylem and (b) is the fallen bark.

[0107] In the figure: C-vascular cambium; Xy-xylem; SC-stone cells; PHE-cork layer; PHL-cork cambium; Pe-cork layer; CO-cortex.

[0108] Example 2

[0109] Preparation of paraffin sections of vascular bundles of cypress with a diameter at breast height of 30 cm and cedar with a diameter at breast height of 50 cm (raw material characteristics: lignified and highly heterogeneous):

[0110] 1) Sampling and fixing steps are the same as in Example 1

[0111] 2) Softening

[0112] The softening treatment was carried out using a combination of hydrochloric acid-ethanol + glycerol-ethanol softening liquid. The hydrochloric acid-ethanol softening liquid was a mixture of 36% concentrated hydrochloric acid and anhydrous ethanol in a volume ratio of 2:3; the glycerol-ethanol softening liquid was a mixture of 70vol% ethanol and glycerol in a volume ratio of 1:1.

[0113] The softening steps are as follows:

[0114] Remove the material from the fixative and place it in a glass bottle. Pour in a hydrochloric acid-ethanol softening solution, just enough to submerge the material and not exceeding half the bottle's volume. Place it in a 55°C oven for 3 hours. After the softening solution treatment is completed, let it stand at room temperature. After the temperature drops to room temperature, pour out the softening solution, pour in 70vol% ethanol, and place it in a 35°C oven to replace the remaining softening solution in the sample. This is done every 2 hours. After two replacements, the third time is left at room temperature overnight. The next day, pour out the 70vol% ethanol and replace it with a glycerol-ethanol softening solution in a 50°C oven for 3 days. After that, place the material in 70vol% ethanol and replace the remaining softening solution in the sample in a 35°C oven. Repeat this three times, each for 2 hours, and the third time is left at room temperature overnight.

[0115] 3)-6) Same as Example 1;

[0116] 7) Slicing, patching and baking

[0117] Before sectioning, prepare a 50% glycerol solution in a 1:1 volume ratio of glycerol to distilled water. Slice the sample on a microtome. After cutting to the desired section, invert the sample and place it in the 50% glycerol solution to soak overnight at room temperature. Use a microtome to cut the wax block into 10μm wax strips. Drop the adhesive film onto the slide and mix well. Gently place the wax strip on the slide and spread it on a slide spreader at 48°C. After the wax strip is fully spread, tilt the slide and absorb the excess adhesive film with absorbent paper. Then, place the slide in a 40°C oven to dry for 24 hours.

[0118] Sections were dewaxed twice in pure xylene for 10 minutes each, then transitioned in 1 / 2 ethanol and 1 / 2 xylene for 3 minutes. Sections were then rehydrated in a sequential order of 3 minutes each in absolute ethanol, 3 minutes in 95% ethanol, 3 minutes in 85% ethanol, 3 minutes in 70% ethanol, and 3 minutes in 50% ethanol. Sections were then stained with safranin for 14 hours. Following safranin staining, sections were dehydrated in a sequential order of 2 minutes each in 50% ethanol, 70% ethanol, 85% ethanol, and 95% ethanol. Sections were then stained with Fast Green for 15 seconds.

[0119] 8) The sealing step is the same as in Example 1.

[0120] Figure 2 Paraffin sections of vascular bundles of cypress with a diameter of 30 cm at breast height. Figure 3 Paraffin sections of cedar vascular bundles (DBH+ 50 cm). Ph - phloem; C - vascular cambium; Xy - xylem.

[0121] Example 3

[0122] Preparation of paraffin sections of one-year-old Masson pine seedling stems (raw material characteristics: moderate degree of lignification and heterogeneity):

[0123] 1) Sampling and fixing steps are the same as in Example 1

[0124] 2) Softening

[0125] The softening treatment was carried out using a combination of hydrochloric acid-ethanol + glycerol-ethanol softening liquid. The hydrochloric acid-ethanol softening liquid was a mixture of 36% concentrated hydrochloric acid and anhydrous ethanol in a volume ratio of 2:3; the glycerol-ethanol softening liquid was a mixture of 70vol% ethanol and glycerol in a volume ratio of 1:1.

[0126] Remove the material from the fixative and place it in a glass bottle. Pour in a hydrochloric acid-ethanol softening solution, just enough to submerge the material and not exceeding half the bottle's volume. Place it in a 55°C oven for 2 hours. After the softening solution treatment is completed, let it stand at room temperature. After the temperature drops to room temperature, pour out the softening solution, pour in 70vol% ethanol, and place it in a 35°C oven to replace the remaining softening solution in the sample. This is done every 2 hours. After two replacements, the third time is left at room temperature overnight. The next day, pour out the 70vol% ethanol and replace it with a glycerol-ethanol softening solution in a 50°C oven for 2 days. After that, place the material in 70vol% ethanol and replace the remaining softening solution in the sample in a 35°C oven. Repeat this three times, each for 2 hours, and the third time is left at room temperature overnight.

[0127] 3)-8) are the same as in Example 2.

[0128] Figure 4 This is a paraffin section of the stem of a one-year-old Masson pine seedling. Ph - phloem; C - vascular cambium; Xy - xylem.

[0129] Comparative Example 1

[0130] Paraffin sections were prepared using the wood core of a Masson pine tree over 30 years old using Zhou Naifu's method for preparing paraffin sections of heterogeneous woody plant tissues:

[0131] 1. The material was fixed with a modified fixative (70% tert-butyl alcohol: 35%–40% formaldehyde: propionic acid: glycerol = 85:5:5:5, v / v / v / v) and vacuumed until the material sank. The fixation was continued for 48 h.

[0132] 2. Place the stem segments in a hydrogen peroxide-glacial acetic acid mixed solution (1:1, v / v), vacuum for 30 minutes, and treat for 3 hours. Then transfer the material to a covered glass bottle filled with a 70% tert-butyl alcohol-propylene glycol mixed solution (1:1, v / v) and place it in a 50°C constant temperature incubator for 2 days.

[0133] 3. Use the tert-butanol gradient dehydration method, i.e., 50% tert-butanol for 1.5 hours → 70% tert-butanol for 1.5 hours → 85% tert-butanol for 1.5 hours → 95% tert-butanol for 1 hour → pure tert-butanol for 30 minutes → pure tert-butanol for 30 minutes. Vacuum treatment is performed after changing the dehydrating agent.

[0134] 4. Immerse the stem segments in a 50% paraffin solution (tert-butyl alcohol: paraffin = 1:1, v / v) for 6 hours at 54–56°C. Then, transfer them to a 75% paraffin solution (tert-butyl alcohol: paraffin = 1:3, v / v) for 6 hours at 56–58°C. Finally, transfer them to pre-melted pure paraffin at 58–60°C. Change the paraffin solution every 4 hours for three times. This entire process should be performed in a constant temperature chamber. After embedding, directly section the sections.

[0135] Figure 5 The paraffin sections prepared in Comparative Example 1 showed that the sections were severely damaged and were not suitable for slicing highly lignified materials.

[0136] Comparative Example 2

[0137] The preparation process is the same as that of Example 1, except that the softening step in step 2) is omitted.

[0138] Figure 6 This is a slice sample prepared in Comparative Example 2. The results show that the slice is broken.

[0139] Comparative Example 3

[0140] The only difference from Example 1 is that no wooden block base is used in the slicing process, and the softening process in step 2) is performed using a single hydrochloric acid-ethanol softening solution: the sample is treated with the hydrochloric acid-ethanol softening solution in a 55°C oven for 8 hours, and then placed in a 35°C oven with 70 vol% ethanol to replace the remaining softening solution in the sample every 2 hours. After replacing it twice, the sample is placed in a room temperature overnight for the third time.

[0141] Figure 7 These sections were prepared without a wooden base and were softened over time. The results show that the thickness of the sections varied due to slight deformation of the embedding box during sectioning, and there was significant damage to the tender areas. Ph - phloem; C - vascular cambium; Xy - xylem.

[0142] Comparative Example 4

[0143] Preparation of paraffin sections using raw materials with a high degree of lignification and heterogeneity:

[0144] 1) Sampling and fixation are the same as in Example 1

[0145] 2) Softening

[0146] The softening treatment was carried out using a combination of hydrochloric acid-ethanol + glycerol-ethanol softening liquid. The hydrochloric acid-ethanol softening liquid was a mixture of 36% concentrated hydrochloric acid and anhydrous ethanol in a volume ratio of 2:3; the glycerol-ethanol softening liquid was a mixture of 70vol% ethanol and glycerol in a volume ratio of 1:1.

[0147] The softening steps are as follows:

[0148] Remove the material from the fixative and place it in a glass bottle. Pour in hydrochloric acid-ethanol softening solution, just enough to submerge the material and not more than half the bottle's volume. Place it in a 55°C oven for 4 hours. After the softening solution treatment is completed, let it stand at room temperature. After the temperature drops to room temperature, pour out the softening solution, pour in 70vol% ethanol, and place it in a 35°C oven to replace the remaining softening solution in the sample. This is done every 2 hours. After replacing it twice, leave it at room temperature overnight for the third time. The next day, pour out the 70vol% ethanol and replace it with glycerol-ethanol softening solution in a 50°C oven for 2 days. Then, place the material in 70vol% ethanol and replace the remaining softening solution in the sample in a 35°C oven. Repeat this three times, each for 2 hours, and leave it at room temperature overnight for the third time.

[0149] 3)-6) Same as Example 1;

[0150] 7) Without the 50 vol% glycerol + 50 vol% distilled water soaking process, the specific steps are as follows:

[0151] Use a microtome to cut the wax block into 10 μm wax strips. Drop the adhesive film onto a glass slide and mix evenly. Gently place the wax strip on the glass slide and spread the slide on a slide spreader at 48°C. After the wax strip is fully spread, tilt the slide and remove the excess adhesive film with absorbent paper. Then, dry the slide in a 40°C oven for 24 hours.

[0152] Sections were dewaxed twice in pure xylene for 10 minutes each, then transitioned in 1 / 2 ethanol and 1 / 2 xylene for 3 minutes. Sections were then rehydrated in a sequential order of anhydrous ethanol (3 minutes twice), 95% ethanol (3 minutes), 85% ethanol (3 minutes), 70% ethanol (3 minutes), and 50% ethanol (3 minutes). Sections were then stained with safranin for 14 hours. Following safranin staining, sections were dehydrated in a sequential order of 50% ethanol, 70% ethanol, 85% ethanol, and 95% ethanol (2 minutes each). Sections were then stained with Fast Green for 15 seconds.

[0153] Figure 8 This is a wood core slice from a Masson pine stem over 30 years old, prepared in Comparative Example 4, that was not soaked in 50 vol% glycerol + 50 vol% distilled water. The results show that this may result in incomplete xylem. Ph = phloem; C = vascular cambium; Xy = xylem.

[0154] Compared with existing methods, the present invention proposes a softening method that uses a combination of two softening solutions: 1. hydrochloric acid-ethanol + glycerol-ethanol, and 2. NaOH-ethanol + glycerol-ethanol. The softening process can be adjusted according to the material and requirements, making it suitable for slicing materials of different hardness levels. Furthermore, compared to traditional slicing methods, the present invention proposes a softening treatment before slicing: after the wax block is cut with a microtome, it is soaked in 50% glycerol. The glycerol gradually penetrates the material contained in the wax block over time, softening the material while further reducing the friction between the material and the blade, significantly improving the slicing effect. Furthermore, a treatment method is proposed in which the wax block is inverted and allowed to stand at 37-40°C in 50% glycerol for at least 10 minutes before slicing, further improving the slicing effect. This method overcomes the problems of cracked, curled, and incomplete slices when making highly lignified and heterogeneous materials with current paraffin wax slice making methods. Experiments have confirmed that the slices produced are structurally complete, clear, and highly applicable.

[0155] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for preparing paraffin sections of highly lignified and highly heterogeneous materials, characterized in that: The following steps are involved: 1) Sampling and fixation After sampling, the material was placed in FAA fixative, vacuumed, and fixed at 4°C for more than 72 hours; 2) Softening The fixed material is placed in a hydrochloric acid-ethanol softening solution or a NaOH-ethanol softening solution for the first softening, and then the softening solution is replaced with 70 vol% ethanol. After the replacement is completed, the material is placed in a glycerol-ethanol softening solution for the second softening; The hydrochloric acid-ethanol softening solution is a mixture of 36% concentrated hydrochloric acid and anhydrous ethanol in a volume ratio of 2:3; The NaOH-ethanol softening solution is a mixture of NaOH and 70 vol% ethanol in a mass volume ratio of 24 g:100 mL; The glycerol-ethanol softening liquid is a mixture of 70 vol% ethanol and glycerol in a volume ratio of 1:1; 3) Dehydration After the softening of the material was completed, it was placed in 70 vol% ethanol for dehydration and softening liquid replacement for 3 times, each time for 2 hours. The first two times were placed in a 35°C oven for treatment, and the last time was placed at room temperature overnight. The next day, the material was dehydrated step by step with an ethanol concentration and time gradient of 70 vol%-2h, 85 vol%-2h, 95 vol%-2h, 100 vol%-1h, and 100 vol%-1h. 4) Transparency The dehydrated material was transitioned in 50 vol% xylene + 50 vol% anhydrous ethanol, then transparentized with xylene, and then placed in a mixed solution of 50 vol% xylene + 50 vol% paraffin at 40°C overnight; 5) Wax dipping The material was wax-impregnated at 60°C; 6) Embedding Embed the material after wax immersion in paraffin wax; 7) Slicing, patching and baking The embedded material was placed in a 50 vol% glycerol + 50 vol% aqueous solution, and then sliced, mounted, baked, and then stained; 8) Sealing process: The stained sections were treated in sequence with 95 vol% ethanol solution, 100 vol% ethanol solution, 50 vol% xylene + 50 vol% anhydrous ethanol, and xylene solution twice, and then sealed with neutral gum. The temperature of the first softening is 55° C., and the time is 1-4 hours; the temperature of the second softening is 50° C., and the time is 2-5 days.

2. The method according to claim 1, characterized in that The first softening time is 2h.

3. The method according to claim 1, characterized in that The second softening time is 2 days.

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