CN114235536B - Slice preparation method for observing lignin deposition of stems of two-year-old masson pine - Google Patents
Slice preparation method for observing lignin deposition of stems of two-year-old masson pine Download PDFInfo
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- 235000011609 Pinus massoniana Nutrition 0.000 title claims abstract description 33
- 241000018650 Pinus massoniana Species 0.000 title claims abstract description 33
- 230000008021 deposition Effects 0.000 title claims abstract description 25
- 229920005610 lignin Polymers 0.000 title claims abstract description 25
- 238000002360 preparation method Methods 0.000 title abstract description 6
- 239000012188 paraffin wax Substances 0.000 claims abstract description 27
- 238000000034 method Methods 0.000 claims abstract description 20
- 238000007789 sealing Methods 0.000 claims abstract description 17
- 230000008569 process Effects 0.000 claims abstract description 10
- 238000004043 dyeing Methods 0.000 claims abstract description 8
- 238000005070 sampling Methods 0.000 claims abstract description 4
- 238000004018 waxing Methods 0.000 claims abstract description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 124
- 239000000243 solution Substances 0.000 claims description 68
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 claims description 47
- 235000019441 ethanol Nutrition 0.000 claims description 40
- 239000001993 wax Substances 0.000 claims description 20
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 15
- 239000011521 glass Substances 0.000 claims description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 15
- 239000008096 xylene Substances 0.000 claims description 15
- 239000011259 mixed solution Substances 0.000 claims description 14
- 238000007598 dipping method Methods 0.000 claims description 13
- 230000018044 dehydration Effects 0.000 claims description 10
- 238000006297 dehydration reaction Methods 0.000 claims description 10
- 239000012153 distilled water Substances 0.000 claims description 9
- 229960000583 acetic acid Drugs 0.000 claims description 7
- OBUOQZXTPAZQNP-UHFFFAOYSA-N butan-1-ol;propane-1,2,3-triol Chemical group CCCCO.OCC(O)CO OBUOQZXTPAZQNP-UHFFFAOYSA-N 0.000 claims description 7
- 239000012362 glacial acetic acid Substances 0.000 claims description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 6
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 claims description 6
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 6
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 claims description 6
- 235000011187 glycerol Nutrition 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 6
- 229910001220 stainless steel Inorganic materials 0.000 claims description 6
- 239000010935 stainless steel Substances 0.000 claims description 6
- 239000000463 material Substances 0.000 claims description 5
- 229910052739 hydrogen Inorganic materials 0.000 claims description 4
- 239000001257 hydrogen Substances 0.000 claims description 4
- 125000004435 hydrogen atom Chemical class [H]* 0.000 claims description 4
- WSFSSNUMVMOOMR-NJFSPNSNSA-N methanone Chemical compound O=[14CH2] WSFSSNUMVMOOMR-NJFSPNSNSA-N 0.000 claims description 4
- 230000007704 transition Effects 0.000 claims description 4
- 108010010803 Gelatin Proteins 0.000 claims description 3
- JPYHHZQJCSQRJY-UHFFFAOYSA-N Phloroglucinol Natural products CCC=CCC=CCC=CCC=CCCCCC(=O)C1=C(O)C=C(O)C=C1O JPYHHZQJCSQRJY-UHFFFAOYSA-N 0.000 claims description 3
- 238000001816 cooling Methods 0.000 claims description 3
- 238000001035 drying Methods 0.000 claims description 3
- 229920000159 gelatin Polymers 0.000 claims description 3
- 239000008273 gelatin Substances 0.000 claims description 3
- 235000019322 gelatine Nutrition 0.000 claims description 3
- 235000011852 gelatine desserts Nutrition 0.000 claims description 3
- QCDYQQDYXPDABM-UHFFFAOYSA-N phloroglucinol Chemical compound OC1=CC(O)=CC(O)=C1 QCDYQQDYXPDABM-UHFFFAOYSA-N 0.000 claims description 3
- 229960001553 phloroglucinol Drugs 0.000 claims description 3
- 238000007712 rapid solidification Methods 0.000 claims description 3
- 238000003892 spreading Methods 0.000 claims description 3
- 230000007480 spreading Effects 0.000 claims description 3
- 238000003860 storage Methods 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- 238000005520 cutting process Methods 0.000 claims description 2
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 claims description 2
- 238000002156 mixing Methods 0.000 claims description 2
- 238000009966 trimming Methods 0.000 claims description 2
- 230000034655 secondary growth Effects 0.000 abstract description 4
- 230000007935 neutral effect Effects 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 2
- 241000218641 Pinaceae Species 0.000 description 2
- 235000005205 Pinus Nutrition 0.000 description 2
- 241000218602 Pinus <genus> Species 0.000 description 2
- 235000011613 Pinus brutia Nutrition 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 241001391944 Commicarpus scandens Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000001000 micrograph Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 239000002023 wood Substances 0.000 description 1
Classifications
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- G—PHYSICS
- 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
- 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
- G01N1/30—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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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Sampling And Sample Adjustment (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention discloses a preparation method of a slice for observing lignin deposition of stems of two-year-old masson pine, which comprises the steps of sampling, fixing, softening, dehydrating, transparent, waxing, embedding, slicing, sticking, baking, dewaxing, dyeing and sealing. According to the technical scheme, compared with the traditional freehand slicing method for observing lignin deposition and the traditional paraffin slicing method for observing the stem tissue structure, the method has higher efficiency and completeness and multifunction, can obtain a slice for completely and clearly observing lignin deposition of the pinus massoniana stems, can simultaneously observe clear and complete stem tissue structure, and lays a foundation for exploring the secondary growth process of the pinus massoniana stems.
Description
Technical Field
The invention relates to the technical field of pinus massoniana stems slicing, in particular to a slice preparation method for observing lignin deposition of pinus massoniana stems in two years.
Background
Pinus massoniana (Pinus massoniana lamb.) is a Pinaceae (Pinaceae) and Pinus (Pinus) arbor, and is mainly distributed in the eastern wet area of subtropical zone in China, and has the characteristics of wide distribution, wide application, high comprehensive utilization degree of whole tree, and the like. The pinus massoniana is a special fast-growing tree species in China, is one of main afforestation tree species in the south, and has important utilization value in the wood processing industry. The observation of lignin deposition on the stems of the two-year-old masson pine can better understand the secondary growth process of the stems of the masson pine, and provide a reference for the cultivation work of large-diameter materials of the masson pine.
Lignin deposition observation usually adopts freehand slicing, but pinus massoniana stems are hard tissues, the freehand slicing is almost difficult to manufacture, and paraffin slicing is a common method for observing plant tissue anatomical structures, and has the characteristics of relatively simple operation, low cost, capability of manufacturing permanent slices and the like. Therefore, the method for completely and clearly observing lignin deposition of the pinus massoniana stems is a problem to be solved, and the combination of paraffin slicing and lignin deposition dyeing can be used for simultaneously and better observing the tissue structure and lignin deposition of the pinus massoniana stems, so that a foundation is laid for exploring the secondary growth process of the pinus massoniana stems.
Disclosure of Invention
Aiming at the problems, the invention aims to provide a slice preparation method for observing lignin deposition of stems of two-year-old pinus massoniana, overcomes the defects of the prior art, and solves the problems that the stems of pinus massoniana are hard in quality, tissue cells are thick in wall, and free-hand slicing, incomplete slicing, unclear, difficult to dye and the like cannot be performed. The slicing method is easy to observe the lignin deposition of the pinus massoniana stems, and solves the problems that the traditional lignin deposition is difficult to observe on the harder woody plants, and the paraffin slice is easy to break and break in the material with higher hardness.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows: a method for preparing a slice for observing lignin deposition from stems of two-year-old masson pine, comprising the following steps:
1) Sampling and fixing
Placing long stem segments with the bottom of about 5mm of the stem of the two-year-old masson pine in FAA fixing solution, and fixing for more than 48 hours at 4 ℃ in a refrigerator;
2) Softening of
Taking out the stem from the FAA fixing solution, washing the fixing solution on the surface of the stem with distilled water, placing the stem in a hydrogen peroxide-glacial acetic acid mixed solution for 5 hours, transferring the stem into a 70% tertiary butanol-glycerol mixed solution, sealing and placing in a 50 ℃ incubator for 7 days, and replacing the 70% tertiary butanol-glycerol mixed solution once a day;
3) Dewatering
The softened stem segments are dehydrated for the first time by 70 percent ethanol, the solution is replaced for a plurality of times during the first dehydration, each time the stem segments are dehydrated for 2 hours in 70 percent ethanol, and then the stem segments are placed in a refrigerator at 4 ℃ for overnight storage; the next day is dehydrated by ethanol with the concentration gradient of 85-100 percent increasing gradually;
4) Transparent and transparent
The dehydrated stem segments are transited in 100 percent dimethylbenzene and absolute ethyl alcohol solution for 2 hours, transparent is carried out twice in 100 percent dimethylbenzene solution after the transition is completed for 2 hours, and the stem segments are placed in 100 percent dimethylbenzene and paraffin solution after the transparent is completed, and are stored overnight in a constant temperature box at 40 ℃;
5) Wax dipping
Placing the transparent stem in a 60 ℃ incubator for wax dipping treatment, wherein pure wax is used in the wax dipping process, and the pure wax is replaced for 6 times during the wax dipping process, wherein the interval time is respectively 4h, 2h and 2h overnight;
6) Embedding
Embedding the stem segment subjected to the final overnight waxing on the next day, specifically pouring the stem segment and paraffin into a stainless steel embedding bottom die together, adjusting the position of a material by using an anatomic needle according to the slicing requirement, attaching a plastic embedding box, pouring some melted paraffin from the upper part of the plastic embedding box, cooling for 5min at normal temperature, placing on ice for rapid solidification, and then removing the stainless steel bottom die to obtain paraffin blocks tightly connected with the plastic embedding box;
7) Slice, patch and roast slice
Trimming the embedded paraffin blocks, cutting into slices of 8-12 mu m by using a slicing machine, transferring the slices onto a glass slide, placing the glass slide on a 40 ℃ spreading machine for 5min, taking up the glass slide, standing until superfluous water is drained, and then placing the glass slide in a 40 ℃ oven for drying for 1d;
8) Dewaxing
Dewaxing the dried slices in 100% xylene solution for 3 times, wherein the treatment time is 3min each time, then transiting in 100% xylene and 100% ethanol solution for 3min, then transferring to 100% ethanol solution for two times, wherein the treatment time is 3min each time, and then immersing in 95% ethanol solution for 3min;
9) Dyeing
Transferring the dewaxed slice to 5% phloroglucinol and 95% ethanol solution for 3min, and then treating with concentrated hydrochloric acid for 1min for dyeing;
10 Sealing sheet)
And transferring the dyed slice to 95% ethanol solution, 100% dimethylbenzene+100% ethanol solution, 100% dimethylbenzene solution and 100% dimethylbenzene solution in turn, performing sealing treatment after each treatment for 3min, wherein the sealing treatment is 100% dimethylbenzene+neutral gum solution, and immediately performing photographing observation treatment after the sealing treatment is finished.
Preferably, in step 1), the FAA fixing solution is a mixed solution of 90ml of 70% ethanol, 5ml of formaldehyde, 5ml of glacial acetic acid and 5ml of glycerol.
Preferably, the volume ratio of the hydrogen peroxide-glacial acetic acid mixed solution and the 70% tertiary butanol-glycerol mixed solution in the step 2) is 1:1.
Preferably, in step 3) dehydration is carried out with three concentration gradients of 85%, 95% and 100% ethanol, the dehydration time of 85% and 95% ethanol being 2h, and the dehydration time of 100% ethanol being 1h each time.
Preferably, in the step 4), the volume ratio of the 100% xylene+absolute ethanol solution to the 100% xylene+paraffin solution is 1:1.
Preferably, in the step 7), 1 drop of the first solution and 2 drops of the second solution are dropped on the glass slide and mixed uniformly before placing the slice, and the first solution comprises the following components: 1.5g gelatin, 5ml glycerin, 2g phenol, 100ml distilled water; the component B is as follows: 4ml of formaldehyde and 100ml of distilled water.
Preferably, in step 8), the solution volume ratio of 100% xylene to 100% ethanol solution is 1:1.
Preferably, in step 10), the solution volume ratios of 100% xylene+100% ethanol solution are 1:1, and the solution volume ratios of 100% xylene+neutral gum solution are 1:2.
The beneficial effects of the invention are as follows: according to the technical scheme, the paraffin section with clear and complete stem tissue structure and clear and easily visible lignin deposition of the two-year-old masson pine can be obtained, and the paraffin section obtained by the section method is easy to observe the stem tissue structure and lignin deposition of the two-year-old masson pine, so that a foundation is laid for exploring the secondary growth process of the stem of the two-year-old masson pine.
Drawings
FIG. 1 is a section of the tissue structure of a two year old pinus massoniana stem according to an embodiment of the invention.
FIG. 2 is a photomicrograph of the present invention looking at lignin deposition from the slice of FIG. 1.
Detailed Description
In order to enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention is further described below with reference to the accompanying drawings and examples.
1-2, a slice preparation method for observing lignin deposition of stems of two-year-old masson pine comprises the following steps:
1) Sampling and fixing
Placing long stem segments with the bottom of about 5mm of the stem of the two-year-old masson pine in FAA fixing solution, and fixing for more than 48 hours at 4 ℃ in a refrigerator; wherein the FAA fixing solution is a mixed solution of 90ml of 70% ethanol, 5ml of formaldehyde, 5ml of glacial acetic acid and 5ml of glycerol.
2) Softening of
Taking out the stem from the FAA fixing solution, washing the fixing solution on the surface of the stem with distilled water, placing the stem in a hydrogen peroxide-glacial acetic acid mixed solution with the concentration of 1:1 and v/v for 5 hours, transferring the stem into a 70% tertiary butanol-glycerol mixed solution with the concentration of 1:1 and v/v, sealing the stem, placing the stem in a 50 ℃ incubator for 7 days, and replacing the 70% tertiary butanol-glycerol mixed solution with the concentration of 1:1 and v/v once a day.
3) Dewatering
The softened stem segments are dehydrated for the first time by 70 percent ethanol, the solution is replaced for a plurality of times (preferably 4 times) during the period, each time, the stem segments are dehydrated for 2 hours in 70 percent ethanol, and then are placed in a refrigerator at 4 ℃ for overnight storage; the next day was dehydrated with three concentration gradients of 85%, 95%, 100% ethanol, the dehydration time of 85% and 95% ethanol was 2 hours, and the dehydration time of 100% ethanol was 1 hour each time.
4) Transparent and transparent
The dehydrated stem segments are transited in 100% dimethylbenzene and absolute ethyl alcohol solution of 1:1 v/v for 2 hours, transparent is carried out twice in 100% dimethylbenzene solution after the transition is completed for 2 hours, and the stem segments are placed in 100% dimethylbenzene and paraffin solution of 1:1 v/v after the transparent is completed, and are stored overnight in a constant temperature box of 40 ℃.
5) Wax dipping
And (3) placing the transparent stem sections in a 60 ℃ incubator for wax dipping treatment, wherein pure wax is used in the wax dipping process, and the pure wax is replaced for 6 times during the wax dipping process, wherein the interval time is respectively 4 hours, 2 hours and overnight.
6) Embedding
Embedding the stem segment after the final overnight wax dipping in the next day, specifically pouring the stem segment and the paraffin into a stainless steel embedding bottom die together, adjusting the position of a material by using an anatomic needle according to the slicing requirement, attaching a plastic embedding box, pouring some melted paraffin from the upper part of the plastic embedding box, cooling for 5min at normal temperature, placing the paraffin on ice for rapid solidification, and then removing the stainless steel bottom die to obtain a paraffin block tightly connected with the plastic embedding box.
7) Slice, patch and roast slice
The embedded paraffin block is trimmed into square or rectangle with parallel upper and lower sides, the square or rectangle is clamped by a slicer (Leica RM2235, germany), the paraffin block is cut into slices with the size of 8-12 mu m, the cut slices naturally form a paraffin tape, and the cut paraffin tape is lightly placed on clean paper by a writing brush. And (3) dripping 1 drop of nail liquid and 2 drops of ethyl liquid at the center of the glass slide by using a rubber head dropper, uniformly mixing, transferring the wax belt onto the glass slide by using forceps and an dissecting needle, placing the glass slide on a 40 ℃ slide spreading machine for about 5 minutes, then taking up the glass slide, standing until superfluous water is drained, and placing the glass slide in a 40 ℃ oven for drying for 1d. Wherein the first liquid comprises the following components: 1.5g gelatin, 5ml glycerin, 2g phenol, 100ml distilled water; the component B is as follows: 4ml of formaldehyde, 100ml of distilled water, which was treated by filtration with filter paper.
8) Dewaxing
The dried slices are subjected to dewaxing treatment for 3 times in 100% xylene solution, wherein the treatment time is 3min each time, then the slices are transited in 1:1 v/v 100% xylene and 100% ethanol solution for 3min, and then the slices are transferred to 100% ethanol solution for two times, wherein the treatment time is 3min each time, and then the slices are soaked in 95% ethanol solution for 3min.
9) Dyeing
The dewaxed sections were transferred to 5% phloroglucinol +95% ethanol solution for 3min, and then stained with concentrated hydrochloric acid for 1 min.
10 Sealing sheet)
The dyed slices are sequentially transferred to 95% ethanol solution, 100% ethanol solution, 1:1, v/v 100% xylene+100% ethanol solution, 100% xylene solution and 100% xylene solution for 3min, then subjected to tablet sealing treatment, wherein the tablet sealing is 1:2, v/v 100% xylene+neutral gum solution, and immediately photographing and observing treatment after the tablet sealing is finished.
Referring to fig. 1-2, fig. 1 is a section of a tissue structure of a two-year-old pinus massoniana stem according to an embodiment of the invention, and fig. 2 is a micrograph of lignin deposition of the section. Wherein Pi is medulla; xy is xylem; ph is phloem; ca is used as a cambium, the color of red dyeing in xylem indicates that lignin deposition exists, the darker the color is, the more lignin deposition is represented, and the paraffin section with clear and complete stem tissue structure and clear and easily visible lignin deposition of the two-year-old masson pine can be obtained by the method, so that the stem tissue structure and lignin deposition of the two-year-old masson pine can be observed better.
The foregoing has shown and described the basic principles, principal features and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and that the above embodiments and descriptions are merely illustrative of the principles of the present invention, and various changes and modifications may be made without departing from the spirit and scope of the invention, which is defined in the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (1)
1. A method for preparing a slice for observing lignin deposition from stems of two-year-old masson pine, comprising the following steps:
1) Sampling and fixing
Placing long stem segments with the length of about 5mm at the bottom of two-year-old masson pine stems into FAA fixing solution, and fixing the FAA fixing solution for more than 48 hours at the temperature of 4 ℃ in a refrigerator, wherein the FAA fixing solution is a mixed solution of 90ml of 70% ethanol, 5ml of formaldehyde, 5ml of glacial acetic acid and 5ml of glycerol;
2) Softening of
Taking out the stem from the FAA fixing solution, washing the fixing solution on the surface of the stem with distilled water, placing the stem in a hydrogen peroxide-glacial acetic acid mixed solution with the concentration of 1:1 and v/v for 5 hours, transferring the stem into a 70% tertiary butanol-glycerol mixed solution with the concentration of 1:1 and v/v, sealing the stem, placing the stem in a 50 ℃ incubator, treating the stem for 7 days, and replacing the stem with the 70% tertiary butanol-glycerol mixed solution with the concentration of 1:1 and v/v once a day;
3) Dewatering
The softened stem segments are dehydrated for the first time by 70 percent ethanol, and the solution is replaced for four times during the first dehydration, each time dehydrated for 2 hours in 70 percent ethanol, and then placed in a refrigerator at 4 ℃ for overnight storage; the next day, ethanol with three concentration gradients of 85%, 95% and 100% is used for dehydration, the dehydration time of the ethanol with the concentration of 85% and 95% is 2 hours, and the dehydration time of the ethanol with the concentration of 100% is 1 hour each time;
4) Transparent and transparent
The dehydrated stem segments are transited in 100% dimethylbenzene and absolute ethyl alcohol solution of 1:1 v/v for 2 hours, the stem segments are transparent in 100% dimethylbenzene solution for 2 hours after the transition, and the stem segments are placed in 100% dimethylbenzene and paraffin solution of 1:1 v/v after the transition, and are stored overnight in a constant temperature box of 40 ℃;
5) Wax dipping
Placing the transparent stem in a 60 ℃ incubator for wax dipping treatment, wherein pure wax is used in the wax dipping process, and the pure wax is replaced for 6 times during the wax dipping process, wherein the interval time is respectively 4h, 2h and 2h overnight;
6) Embedding
Embedding the stem segment subjected to the final overnight waxing on the next day, specifically pouring the stem segment and paraffin into a stainless steel embedding bottom die together, adjusting the position of a material by using an anatomic needle according to the slicing requirement, attaching a plastic embedding box, pouring some melted paraffin from the upper part of the plastic embedding box, cooling for 5min at normal temperature, placing on ice for rapid solidification, and then removing the stainless steel bottom die to obtain paraffin blocks tightly connected with the plastic embedding box;
7) Slice, patch and roast slice
Trimming the embedded paraffin blocks, cutting into slices of 8-12 mu m by using a slicing machine, transferring the slices onto a glass slide, standing the glass slide on a 40 ℃ spreading machine for 5min, taking the glass slide, standing until superfluous water is drained, then drying the glass slide in a 40 ℃ oven for 1d, and dripping 1 drop of nail liquid and 2 drops of ethyl liquid on the glass slide and uniformly mixing before placing the slices, wherein the nail liquid comprises the following components: 1.5g gelatin, 5ml glycerin, 2g phenol, 100ml distilled water; the component B is as follows: 4ml of formaldehyde and 100ml of distilled water;
8) Dewaxing
Dewaxing the dried slices in 100% xylene solution for 3 times, wherein the time of each treatment is 3min, then transiting in 1:1 v/v 100% xylene and 100% ethanol solution for 3min, transferring to 100% ethanol solution for two times, wherein the time of each treatment is 3min, and then immersing in 95% ethanol solution for 3min;
9) Dyeing
Transferring the dewaxed slice to 5% phloroglucinol and 95% ethanol solution for 3min, and then treating with concentrated hydrochloric acid for 1min for dyeing;
10 Sealing sheet)
And transferring the dyed slice to 95% ethanol solution, 100% dimethylbenzene+100% ethanol solution, 100% dimethylbenzene solution and 100% dimethylbenzene solution in turn, performing sealing treatment after each treatment for 3min, wherein the sealing treatment is 1:2, and the sealing treatment is performed immediately after the sealing treatment is finished.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103994906A (en) * | 2014-04-30 | 2014-08-20 | 河南科技大学 | Making method of wood cross section year-by-year growth ring microscopic slices |
| CN106525530A (en) * | 2016-11-04 | 2017-03-22 | 河南科技大学 | Paraffin slicing method of tree stem tissue |
| CN107167350A (en) * | 2017-05-16 | 2017-09-15 | 云南省农业科学院园艺作物研究所 | A kind of preparation method of the paraffin section of eggplant rhizome portion tissue |
| CN107655731A (en) * | 2017-02-08 | 2018-02-02 | 云南省农业科学院花卉研究所 | For the larger explant leaf bud of carnation and the paraffin section method of bud |
| CN108333013A (en) * | 2018-02-10 | 2018-07-27 | 北京林业大学 | A kind of preparation method of jujube flower paraffin section |
| CN110793829A (en) * | 2019-11-22 | 2020-02-14 | 西藏自治区农牧科学院农业资源与环境研究所 | Method for slicing highland barley stem profile paraffin suitable for plateau environment |
| CN110954384A (en) * | 2019-12-16 | 2020-04-03 | 贵州大学 | Preparation method of masson pine resin tract paraffin section |
-
2021
- 2021-12-24 CN CN202111595314.7A patent/CN114235536B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103994906A (en) * | 2014-04-30 | 2014-08-20 | 河南科技大学 | Making method of wood cross section year-by-year growth ring microscopic slices |
| CN106525530A (en) * | 2016-11-04 | 2017-03-22 | 河南科技大学 | Paraffin slicing method of tree stem tissue |
| CN107655731A (en) * | 2017-02-08 | 2018-02-02 | 云南省农业科学院花卉研究所 | For the larger explant leaf bud of carnation and the paraffin section method of bud |
| CN107167350A (en) * | 2017-05-16 | 2017-09-15 | 云南省农业科学院园艺作物研究所 | A kind of preparation method of the paraffin section of eggplant rhizome portion tissue |
| CN108333013A (en) * | 2018-02-10 | 2018-07-27 | 北京林业大学 | A kind of preparation method of jujube flower paraffin section |
| CN110793829A (en) * | 2019-11-22 | 2020-02-14 | 西藏自治区农牧科学院农业资源与环境研究所 | Method for slicing highland barley stem profile paraffin suitable for plateau environment |
| CN110954384A (en) * | 2019-12-16 | 2020-04-03 | 贵州大学 | Preparation method of masson pine resin tract paraffin section |
Non-Patent Citations (1)
| Title |
|---|
| 王厚麟.广州及周边地区维管植物.广东高等教育出版社,2012,第177页-第181页. * |
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