A method for preparing paraffin sections of Amomum villosum rhizome

Abstract

The invention discloses a paraffin section preparation method of Amomum villosum rhizome, comprising the following steps: (1) collecting materials; (2) fixing; (3) dehydrating and transparentizing; (4) performing wax dipping and embedding on the dehydrated materials in the following steps in sequence: (5) slicing the obtained wax block to obtain a wax strip; (6) transferring the cut wax strip to water, unfolding it on the water surface, and then placing it on a clean glass slide, marking it or attaching a label to it and placing it on a slicing rack to obtain the unfolded material; (7) staining; (8) sealing the slide with neutral gum to obtain the paraffin section of Amomum villosum rhizome. The method provided by the invention ensures the slicing effect, and can cut out sections with complete and clear tissue structure and clear and distinct staining, so as to facilitate the observation of the microstructure of Amomum villosum rhizome, and can provide reliable technical support for the anatomical research of Amomum villosum rhizome and the revelation of the relationship and mechanism between functional traits.

Description

Technical Field

[0001] The invention relates to the technical field of paraffin sections, in particular to a method for preparing paraffin sections of Amomum villosum rhizomes. Background Art

[0002] Amomum villosum Lour. is a perennial shade-tolerant evergreen herbaceous medicinal plant of the genus Amomum L. of the Zingiberaceae family. It is native to tropical and subtropical regions and is widely planted in Yunnan, Guangxi and Guangdong in my country. The fruit it produces is one of my country's traditional "four major" southern medicinal varieties, with the effects of removing dampness and stimulating appetite, warming the spleen and stopping diarrhea, regulating qi and stabilizing the fetus. It has a long history of medicinal use and extremely high medicinal value.

[0003] The rhizome of Amomum villosum is the part of the plant where the roots grow, the flowering and fruiting occurs, and it is also the hub for the transportation of water, nutrients and metabolites. It is the core organ of the plant and determines the population growth and yield characteristics of Amomum villosum. However, the research on the rhizome of Amomum villosum has not received much attention, and has only been involved in the medicinal chemical components. Little is known about its morphology and microstructural characteristics, which restricts the research on the growth and development of the plant. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a method for preparing paraffin sections of Amomum villosum rhizomes, which can ensure that the tissue structure of the obtained sections is clear and complete, and is convenient for observing the microstructure of the Amomum villosum rhizomes.

[0005] To achieve the above object, the technical solution of the present invention is as follows:

[0006] A method for preparing paraffin sections of Amomum villosum rhizomes comprises the following steps:

[0007] (1) Material collection: Select the rhizome of Amomum villosum, clean it, and cut it into small sections to obtain cut materials;

[0008] (2) Fixation: Fixing the cut material with FAA fixative to obtain a fixed material; the concentration of the FAA fixative is 65-75%, preferably 70%;

[0009] Wherein, when the rhizomes of Amomum villosum are young rhizomes in step (1), in step (2), the fixation time of the FAA fixative is 45 to 50 hours, preferably 48 hours, and the FAA fixative is replaced once in the middle. Specifically, the fixative is firstly fixed in the FAA fixative for 24 hours, the first fixative is discarded, and then the newly configured fixative is added and fixed for 24 hours;

[0010] When the rhizome of Amomum villosum in step (1) is an old rhizome, in step (2), the FAA fixative is fixed for 20 to 28 hours, preferably 24 hours, and after fixation, the rhizome is transferred to a mixture of anhydrous ethanol and glycerol for softening for 5 to 7 days, wherein the volume ratio of anhydrous ethanol to glycerol is 1:1;

[0011] (3) Dehydration and transparency: gradually increasing the volume concentration of the alcohol solution to dehydrate the fixed material to obtain a dehydrated material, wherein the volume concentration of the alcohol solution is as low as 50% and as high as 100%;

[0012] (4) The dehydrated material is wax-impregnated and embedded using the following steps:

[0013] (4.1) Place in a container filled with paraffin and xylene in a volume ratio of 3:1 and place in an oven at 58-62°C for 40 min-1 h;

[0014] (4.2) Transfer to a container containing paraffin and xylene in a volume ratio of 2:1 and place in an oven at 58-62°C for 40 min-1 h;

[0015] (4.3) Transfer to a container filled with pure xylene and place in an oven at 58-62°C for 40 min-1 h;

[0016] (4.4) Transfer to a container filled with pure paraffin wax and place in an oven at 58-62°C for 10-14 hours;

[0017] (4.5) Pour the melted paraffin into a disposable embedding box, use tweezers to move the material treated in step (4.2) into the melted paraffin, cool and solidify at room temperature to obtain a wax block;

[0018] (5) slicing the obtained wax block to obtain a wax strip;

[0019] (6) Transfer the cut wax tape into water, unfold it on the water surface, and then place it on a clean glass slide, mark it or label it and place it on a slice rack to obtain the unfolded material;

[0020] (7) Staining: The developed material is first placed in xylene for 15-30 min, then placed in a mixture of alcohol and xylene in a volume ratio of 1:1, 100% alcohol, 95% alcohol, 80% alcohol, 70% alcohol, and 50% alcohol, each soaked for 2 min-3 min; then placed in toluidine blue for 30 s to 50 s; then placed in water for washing for 3 min-5 min; then placed in 50% alcohol, 70% alcohol, 80% alcohol, 95% alcohol, 100% alcohol, and 100% alcohol, each soaked for 2 min-3 min; finally, placed in xylene for 10-20 min;

[0021] (8) Use neutral gum to seal the slides and obtain paraffin sections of Amomum villosum rhizomes.

[0022] Furthermore, when the rhizome of Amomum villosum in step (1) is a young rhizome, in step (3), the dehydration is carried out in the following order: dehydration with 50% ethanol for 1 hour, dehydration with 75% ethanol for 1 hour, dehydration with 80% ethanol for 1 hour, dehydration with 95% ethanol for 1 hour, and dehydration with anhydrous ethanol for 2 hours;

[0023] When the Amomum villosum rhizomes in step (1) are old rhizomes, in step (3), the dehydration is carried out in the following order: 50% ethanol dehydration for 1.5 h, 75% ethanol dehydration for 1.5 h, 80% ethanol dehydration for 1.5 h, 95% ethanol dehydration for 1.5 h, and anhydrous ethanol dehydration for 2 h.

[0024] Furthermore, in steps (4.1) to (4.4), the temperature of the oven is set to 60°C; in steps (4.1) to (4.3), the treatment time of the oven is 1 hour; in step (4.4), the treatment time of the oven is 12 hours.

[0025] Furthermore, in step (5), the thickness of the wax strip is 10-15 um.

[0026] Furthermore, in step (6), the temperature of water is 40-55°C.

[0027] Furthermore, in step (7), the developed material is first placed in xylene for treatment for 15 minutes, then placed in new xylene for treatment for 15 minutes, and then placed in a mixture of alcohol and xylene in a volume ratio of 1:1, 100% alcohol, 95% alcohol, 80% alcohol, 70% alcohol, and 50% alcohol, each soaked for 2 minutes; then placed in toluidine blue for 40 seconds; then placed in water for washing for 2 minutes, and then placed in new water for washing for 2 minutes; then placed in 50% alcohol, 70% alcohol, 80% alcohol, 95% alcohol, 100% alcohol, and 100% alcohol, each soaked for 2 minutes-3 minutes; finally, first soaked in xylene for 10 minutes, and then soaked in new xylene for 10 minutes.

[0028] The beneficial effects of the present invention include at least:

[0029] The present invention uses a specific concentration of FAA fixative to preliminarily treat the rhizome and then uses a mixed solution of anhydrous ethanol and propylene glycol in a mixed volume ratio of 1:1 to soften the old rhizome, thereby preventing the slices from being wrinkled or damaged, and by adjusting the time of subsequent dehydration, wax immersion, staining and other steps and the temperature in some steps, etc., the slicing effect is guaranteed, and slices with complete and clear tissue structure and clear staining can be cut out, thereby facilitating the observation of the microstructure of the Amomum villosum rhizome, and providing reliable technical support for the anatomical research of the Amomum villosum rhizome and the revelation of the relationship and mechanism between functional traits.

[0030] That is, the present invention can improve the quality and clarity of slices, better display the cell structure and tissue morphology of Amomum villosum rhizomes; reduce the possibility of tissue damage and degeneration, maintain the integrity and accuracy of samples; improve the efficiency and accuracy of observation and analysis research, and provide more accurate data and information for further research. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 These are the microscopic structures of the young stem rhizomes of Amomum villosum, where A is a panoramic view of the young stem and B is a partial view of the young stem.

[0032] Figure 2 These are the microscopic structures of the old rhizomes of Amomum villosum, where A is a panoramic view of the old stem and B is a partial view of the old stem.

[0033] Figure 3 These are diagrams of the rhizome structure of Amomum villosum after different dehydration methods, where A: traditional dehydration method; B: improved dehydration method.

[0034] Figure 4 Wax tapes of different thicknesses.

[0035] Figure 5 These are the dyeing effects of different dyes; A: safranin-fast green dyeing; B: toluidine blue dyeing. DETAILED DESCRIPTION

[0036] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0037] In addition, the technical solutions between the various embodiments of the present invention can be combined with each other, but it must be based on the fact that ordinary technicians in the field can implement it. When the combination of technical solutions is contradictory or cannot be implemented, it should be deemed that such combination of technical solutions does not exist and is not within the scope of protection required by the present invention.

[0038] The solution proposed by the present invention is specifically described below through specific embodiments:

[0039] Example 1 for young stems

[0040] Steps for making paraffin sections of Amomum villosum rhizomes:

[0041] 1. Draw materials

[0042] Select young and tender rhizomes of Amomum villosum, clean them, and cut the material into small sections of 2-3 mm.

[0043] 2. Fixation

[0044] The cut young rhizomes (referred to as 'young stems') were fixed with 70% FAA fixative solution by volume for 48 hours, specifically, first fixed in 70% FAA fixative solution for 24 hours, the first fixative solution was poured out, and then the newly prepared 70% FAA fixative solution was added and fixed for 24 hours.

[0045] 3. Dehydration and transparency

[0046] Dehydrate the fixed Amomum villosum rhizome material in the following steps:

[0047] Young stems: 50% ethanol for 1 h → 75% ethanol for 1 h → 80% ethanol for 1 h → 95% ethanol for 1 h → anhydrous ethanol (I) for 1 h → anhydrous ethanol (II) for 1 h (all concentrations are volume percentages).

[0048] Transparency: After dehydration, make it transparent according to the steps of 1 / 2 alcohol + 1 / 2 xylene 1h → pure xylene 1h → pure xylene 1h.

[0049] 4. Wax dipping and embedding

[0050] The transparent Amomum villosum rhizome material is wax-impregnated and embedded in the following steps:

[0051] Paraffin and xylene (volume ratio 3:1) were treated in an oven at 60℃ for 1h → paraffin and xylene (volume ratio 2:1) were treated in an oven at 60℃ for 1h → pure xylene was treated in an oven at 60℃ for 1h → pure paraffin was treated in an oven at 60℃ for 12h to obtain transparent Amomum villosum stem material. Pour the melted paraffin into a disposable embedding box, use tweezers to move the transparent Amomum villosum material into the melted paraffin, cool and solidify at room temperature to obtain a wax block.

[0052] 5. Trimming and slicing

[0053] The paraffin-embedded material was trimmed into a rectangle and sliced ​​to obtain a wax strip with a thickness of 12 μm.

[0054] 6. Spreading and sticking

[0055] Transfer the cut wax strips to warm water with a brush, spread them out on the water surface, place them on a clean glass slide, mark or label them, and place them on a slice rack.

[0056] 7. Dyeing

[0057] Toluidine blue staining was used, and the specific steps were as follows:

[0058] Xylene (Ⅰ) 15min→Xylene transparent 15min(Ⅱ)→Alcohol:xylene (1:1) 2min 2min→100% alcohol 2min→95% alcohol 2min→80% alcohol 2min→70% alcohol 2min→50% alcohol 2min→Toluidine blue 40s→Water wash 2min→Water wash 2min→50% alcohol to remove floating color→70% alcohol 2min→80% alcohol 2min→95% alcohol 2min→100% alcohol 2min→100% alcohol 2min→Alcohol:xylene (1:1) 2min→Xylene transparent 10min→Xylene transparent 10min.

[0059] 8. Seal the film

[0060] Neutral gum was used to seal the sections and paraffin sections of Amomum villosum rhizomes were obtained.

[0061] 9. Observation

[0062] Observation and photography under NIKON 80i biological microscope.

[0063] The slices prepared in this example are as follows Figure 1 shown.

[0064] Example 2: Old stems

[0065] 1. Draw materials

[0066] Select old rhizomes of Amomum villosum, clean them, and cut the material into small sections of 2-3 mm.

[0067] 2. Fixation

[0068] The cut old rhizomes (referred to as 'old stems') were fixed with 70% FAA fixative for 24 h and then transferred to a mixture of anhydrous ethanol and glycerol (volume ratio 1:1) to soften for 6 days.

[0069] 3. Dehydration and transparency

[0070] Dehydrate the fixed Amomum villosum rhizome material in the following steps:

[0071] Old stems: 50% ethanol for 1.5h → 75% ethanol for 1.5h → 80% ethanol for 1.5h → 95% ethanol for 1.5h → anhydrous ethanol (I) for 1h → anhydrous ethanol (II) for 1h (all concentrations are volume percentages).

[0072] 4. Wax dipping and embedding

[0073] The transparent rhizome material of Amomum villosum is wax-impregnated and embedded in the following steps:

[0074] Paraffin and xylene (volume ratio 3:1) were treated in an oven at 60℃ for 1h → paraffin and xylene (volume ratio 2:1) were treated in an oven at 60℃ for 1h → pure xylene was treated in an oven at 60℃ for 1h → pure paraffin was treated in an oven at 60℃ for 12h to obtain transparent Amomum villosum stem material. Pour the melted paraffin into a disposable embedding box, use tweezers to move the transparent Amomum villosum material into the melted paraffin, cool and solidify at room temperature to obtain a wax block.

[0075] 5. Trimming and slicing

[0076] The paraffin-embedded material was trimmed into a rectangle and sliced ​​to obtain a wax strip with a thickness of 12 μm.

[0077] 6. Spreading and sticking

[0078] Transfer the cut wax strips to warm water with a brush, spread them out on the water surface, place them on a clean glass slide, mark or label them, and place them on a slice rack.

[0079] 7. Dyeing

[0080] Toluidine blue staining was used, and the specific steps were as follows:

[0081] Xylene (Ⅰ) 15min→Xylene transparent 15min(Ⅱ)→Alcohol:xylene (1:1) 2min2min→100% alcohol 2min→95% alcohol 2min→80% alcohol 2min→70% alcohol 2min→50% alcohol 2min→Toluidine blue 40s→Water wash 2min→Water wash 2min→50% alcohol to wash away floating color→70% alcohol 2min→80% alcohol 2min→95% alcohol 2min→100% alcohol 2min→100% alcohol 2min→Alcohol:xylene (1:1) 2min→Xylene transparent 10min→Xylene transparent 10min.

[0082] 8. Seal the film

[0083] Neutral gum was used to seal the sections and paraffin sections of Amomum villosum rhizomes were obtained.

[0084] 9. Observation

[0085] Observation and photography under NIKON 80i biological microscope.

[0086] The slices prepared in this example are as follows Figure 2 shown.

[0087] Comparative Example 1

[0088] The difference between this embodiment and embodiment 2 is that step 2 is adjusted to: put into 70% FAA fixative (i.e. 90 mL of 70% ethanol, 5 mL of glacial acetic acid, 5 mL of formaldehyde), and store at 4° C. for 24 hours.

[0089] The rest remains unchanged.

[0090] In this embodiment, since the tissue was not transferred to a mixture of anhydrous ethanol and glycerol (volume ratio 1:1) for softening, the tissue was easily broken in the subsequent steps and a complete slice could not be cut out.

[0091] Comparative Example 2

[0092] Table 1 Effect of different dehydration gradients on slices of Amomum villosum stems

[0093]

[0094] Table 2 Effect of different dehydration gradients on the slice effect of old stems of Amomum villosum

[0095]

[0096] Specific results such as Figure 3 As shown, A is the structure diagram of the rhizome of Amomum villosum numbered 3 in Table 1 (i.e., the traditional dehydration method), and B is the structure diagram of the rhizome of Amomum villosum numbered 1 in Table 1.

[0097] Comparative Example 3 Comparison of different wax tape thicknesses

[0098] Compared with Example 1, this embodiment is the same except that the thickness of the wax strip in step 5 is different. That is, the thickness of the wax strip is adjusted for comparison.

[0099] The results are as follows Figure 4 As shown, there are wax strips with thicknesses of 8 μm, 10 μm and 12 μm respectively. It can be seen that the wax strip with a thickness of 12 μm has better uniformity and clarity.

[0100] Comparative Example 4

[0101] The difference between this embodiment and embodiment 2 is that in step 7, toluidine blue is adjusted to safranin-fast green for dyeing. Figure 5 As shown in A. It can be seen that the staining with safranin-fast green dye ( Figure 5 A) Uneven, stained with toluidine blue stain ( Figure 5 B) The cell structure of the rhizome of Amomum villosum is clear.

[0102] In summary, the present invention improves the fixation, dehydration time, slice thickness, and staining method based on the characteristics of Amomum villosum rhizomes on the basis of traditional paraffin sections, and explores a suitable slicing method for the material. Overall, it has the following effects:

[0103] 1) The quality and clarity of the slices were improved to better display the cell structure and tissue morphology of the rhizome of Amomum villosum.

[0104] 2) Reduces the possibility of tissue damage and degeneration, maintaining sample integrity and accuracy.

[0105] 3) Improve the efficiency and accuracy of observation and analysis research, and provide more accurate data and information for further research.

[0106] It should be noted that, in this article, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the sentence "comprises a ..." does not exclude the existence of other identical elements in the process, method, article or device including the element.

[0107] The serial numbers of the above embodiments of the present invention are only for description and do not represent the advantages or disadvantages of the embodiments.

[0108] The above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. A method for preparing paraffin sections of Amomum villosum rhizomes, characterized in that: The following steps are involved: (1) Material collection: Select the rhizome of Amomum villosum, clean it, and cut it into small sections to obtain cut materials; (2) Fixation: Fixing the cut material with FAA fixative to obtain a fixed material; the concentration of the FAA fixative is 65-75%; Wherein, when the rhizome of Amomum villosum is a young rhizome in step (1), in step (2), the FAA fixative is fixed for 45 to 50 hours, and the FAA fixative is replaced once in the middle; When the rhizome of Amomum villosum in step (1) is an old rhizome, in step (2), the FAA fixative is fixed for 20 to 28 hours, and after fixation, the rhizome is transferred to a mixture of anhydrous ethanol and glycerol for softening for 5 to 7 days, wherein the volume ratio of anhydrous ethanol to glycerol is 1:1; (3) Dehydration and transparency: gradually increase the volume concentration of the alcohol solution to dehydrate the fixed material to obtain a dehydrated material, wherein the volume concentration of the alcohol solution is as low as 50% and as high as 100%; (4) The dehydrated material is wax-impregnated and embedded using the following steps: (4.1) Place in a container filled with paraffin and xylene in a volume ratio of 3:1 and place in an oven at 58~62℃ for 40min-1h; (4.2) Transfer to a container containing paraffin and xylene in a volume ratio of 2:1 and place in an oven at 58~62℃ for 40min-1h; (4.3) Transfer to a container filled with pure xylene and place in an oven at 58-62°C for 40 min-1 h; (4.4) Transfer to a container filled with pure paraffin wax and place in an oven at 58-62℃ for 10-14h; (4.5) Pour the melted paraffin into a disposable embedding box, use tweezers to move the material processed in step (4.4) into the melted paraffin, cool and solidify at room temperature to obtain a wax block; (5) Slicing the obtained wax block to obtain a wax strip; the thickness of the wax strip is 10-15 μm; (6) Transfer the cut wax strips into water, unfold them on the water surface, and then place them on a clean glass slide. Mark or label them and place them on a slice rack to obtain the unfolded material. (7) Staining: The developed material is first placed in xylene for 15-30 minutes, then placed in a mixture of alcohol and xylene in a volume ratio of 1:1, 100% alcohol, 95% alcohol, 80% alcohol, 70% alcohol, and 50% alcohol, and soaked for 2 minutes to 3 minutes respectively; then soaked in toluidine blue for 30 seconds to 50 seconds; then washed in water for 3 minutes to 5 minutes; then soaked in 50% alcohol, 70% alcohol, 80% alcohol, 95% alcohol, 100% alcohol, and 100% alcohol, and soaked for 2 minutes to 3 minutes respectively; then soaked in a mixture of alcohol and xylene in a volume ratio of 1:1 for 2 minutes; finally soaked in xylene for 10-20 minutes; (8) Use neutral gum to seal the slides and obtain paraffin sections of Amomum villosum rhizomes.

2. The method according to claim 1, characterized in that: When the rhizome of Amomum villosum in step (1) is a young rhizome, in step (3), the dehydration is carried out in the following order: dehydration with 50% ethanol for 1 h, dehydration with 75% ethanol for 1 h, dehydration with 80% ethanol for 1 h, dehydration with 95% ethanol for 1 h, and dehydration with anhydrous ethanol for 2 h; When the rhizomes of Amomum villosum in step (1) are old rhizomes, in step (3), the dehydration is carried out in the following order: dehydration with 50% ethanol for 1.5 h, dehydration with 75% ethanol for 1.5 h, dehydration with 80% ethanol for 1.5 h, dehydration with 95% ethanol for 1.5 h, and dehydration with anhydrous ethanol for 2 h.

3. The method according to claim 1, characterized in that: In steps (4.1) to (4.4), the temperature of the oven is set to 60°C; in steps (4.1) to (4.3), the treatment time of the oven is 1 hour; in step (4.4), the treatment time of the oven is 12 hours.

4. The method according to claim 1, characterized in that: In step (6), the temperature of the water is 40-55°C.

5. The method according to claim 1, characterized in that: In step (7), the developed material is first placed in xylene for treatment for 15 minutes, then placed in new xylene for treatment for 15 minutes, and then placed in a mixture of alcohol and xylene in a volume ratio of 1:1, 100% alcohol, 95% alcohol, 80% alcohol, 70% alcohol, and 50% alcohol in turn for 2 minutes each; then placed in toluidine blue for 40 seconds; then placed in water for washing for 2 minutes, and then placed in new water for washing for 2 minutes; then placed in 50% alcohol, 70% alcohol, 80% alcohol, 95% alcohol, 100% alcohol, and 100% alcohol in turn for 2 minutes-3 minutes each; then placed in a mixture of alcohol and xylene in a volume ratio of 1:1 for 2 minutes; finally, first soaked in xylene for 10 minutes, and then soaked in new xylene for 10 minutes.

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