CN116840020A - Preparation method of paraffin section of soft and hard phase plant tissue - Google Patents
Preparation method of paraffin section of soft and hard phase plant tissue Download PDFInfo
- Publication number
- CN116840020A CN116840020A CN202311101420.4A CN202311101420A CN116840020A CN 116840020 A CN116840020 A CN 116840020A CN 202311101420 A CN202311101420 A CN 202311101420A CN 116840020 A CN116840020 A CN 116840020A
- Authority
- CN
- China
- Prior art keywords
- alcohol
- plant tissue
- seconds
- softening
- paraffin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 239000012188 paraffin wax Substances 0.000 title claims abstract description 80
- 238000002360 preparation method Methods 0.000 title claims abstract description 34
- 238000000034 method Methods 0.000 claims abstract description 29
- 230000018044 dehydration Effects 0.000 claims abstract description 14
- 238000006297 dehydration reaction Methods 0.000 claims abstract description 14
- 238000004043 dyeing Methods 0.000 claims abstract description 12
- 238000007789 sealing Methods 0.000 claims abstract description 10
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 73
- 241000196324 Embryophyta Species 0.000 claims description 40
- KRHYYFGTRYWZRS-UHFFFAOYSA-N Fluorane Chemical compound F KRHYYFGTRYWZRS-UHFFFAOYSA-N 0.000 claims description 34
- 239000001993 wax Substances 0.000 claims description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 16
- 239000008096 xylene Substances 0.000 claims description 15
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 claims description 13
- 244000127818 Phalaenopsis amabilis Species 0.000 claims description 13
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 claims description 9
- 238000007598 dipping method Methods 0.000 claims description 5
- 239000001046 green dye Substances 0.000 claims description 5
- 239000001011 safranin dye Substances 0.000 claims description 5
- 239000007787 solid Substances 0.000 claims description 5
- 241001505935 Phalaenopsis Species 0.000 claims description 4
- 230000007935 neutral effect Effects 0.000 claims description 4
- 238000005406 washing Methods 0.000 claims description 4
- 239000011347 resin Substances 0.000 claims description 3
- 229920005989 resin Polymers 0.000 claims description 3
- 238000007667 floating Methods 0.000 claims description 2
- 210000000056 organ Anatomy 0.000 claims description 2
- 238000005086 pumping Methods 0.000 claims description 2
- 150000003738 xylenes Chemical class 0.000 claims description 2
- 239000007788 liquid Substances 0.000 abstract description 5
- OARRHUQTFTUEOS-UHFFFAOYSA-N safranin Chemical compound [Cl-].C=12C=C(N)C(C)=CC2=NC2=CC(C)=C(N)C=C2[N+]=1C1=CC=CC=C1 OARRHUQTFTUEOS-UHFFFAOYSA-N 0.000 abstract description 2
- 238000003892 spreading Methods 0.000 abstract description 2
- 230000007480 spreading Effects 0.000 abstract description 2
- 238000004018 waxing Methods 0.000 abstract 1
- 210000001519 tissue Anatomy 0.000 description 52
- 230000000052 comparative effect Effects 0.000 description 23
- 239000000463 material Substances 0.000 description 21
- 239000000243 solution Substances 0.000 description 16
- 230000000694 effects Effects 0.000 description 11
- 230000008569 process Effects 0.000 description 7
- 239000000203 mixture Substances 0.000 description 6
- 238000001035 drying Methods 0.000 description 5
- 239000000834 fixative Substances 0.000 description 5
- 238000011160 research Methods 0.000 description 5
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 239000011259 mixed solution Substances 0.000 description 3
- 230000000877 morphologic effect Effects 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 2
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 2
- 241000233855 Orchidaceae Species 0.000 description 2
- 238000004140 cleaning Methods 0.000 description 2
- 239000006059 cover glass Substances 0.000 description 2
- 238000005520 cutting process Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 238000009966 trimming Methods 0.000 description 2
- RZSYLLSAWYUBPE-UHFFFAOYSA-L Fast green FCF Chemical compound [Na+].[Na+].C=1C=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C(=CC(O)=CC=2)S([O-])(=O)=O)C=CC=1N(CC)CC1=CC=CC(S([O-])(=O)=O)=C1 RZSYLLSAWYUBPE-UHFFFAOYSA-L 0.000 description 1
- 240000000161 Lagerstroemia indica Species 0.000 description 1
- 235000000283 Lagerstroemia parviflora Nutrition 0.000 description 1
- 239000004902 Softening Agent Substances 0.000 description 1
- 229960000583 acetic acid Drugs 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000000861 blow drying Methods 0.000 description 1
- 230000005200 bud stage Effects 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000004744 fabric Substances 0.000 description 1
- 239000003517 fume Substances 0.000 description 1
- 230000035784 germination Effects 0.000 description 1
- 239000012362 glacial acetic acid Substances 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 238000003475 lamination Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000000149 penetrating effect Effects 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 210000004872 soft tissue Anatomy 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000009489 vacuum treatment Methods 0.000 description 1
- 239000011800 void material Substances 0.000 description 1
Classifications
-
- 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
- G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
-
- 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
-
- 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
- G01N1/36—Embedding or analogous mounting of samples
-
- 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
- G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
- G01N2001/2873—Cutting or cleaving
Abstract
The invention discloses a preparation method of a soft and hard phase plant tissue paraffin section, and relates to the technical field of plant tissue sections. The preparation method of the paraffin section comprises the steps of fixing, softening, dehydrating, waxing, embedding, section and spreading, dewaxing, rehydration, safranine solid-green dyeing and sealing. The preparation method of the paraffin section is improved, and specifically comprises the steps of softening liquid, concentration, softening time, dehydration and the like, so that the method ensures that the method has less damage to tender axillary bud cell tissues and can meet the softening requirement of a lignified and harder main stem part when the preparation method is used for preparing the soft and hard phase plant tissue paraffin section.
Description
Technical Field
The invention relates to the technical field of plant tissue sections, in particular to a preparation method of a soft and hard phase plant tissue paraffin section.
Background
Butterfly orchid @Phalaenopsis) Is of the genus Phalaenopsis of the family OrchidaceaePhalaenosis) The plant is a perennial herb plant, one of the most economic flower tissues of the butterfly orchid is a pedicel, and the pedicel is formed by sprouting and elongating from the latent bud point of the basal part of the leaf where the stem of the butterfly orchid grows.
Paraffin sectioning is a method for observing cell morphology under a microscope, and generally comprises the steps of drawing materials, fixing, dehydrating, transparentizing, penetrating wax, embedding, sectioning, attaching, staining, transparentizing, sealing and the like, and paraffin sectioning can be used for researching plant microstructure. Researchers often need to observe microscopic changes in the developmental process of germination, growth of the butterfly orchid pedicel from the axillary bud stage by paraffin section.
Plant paraffin section has few reports about the section of flower buds, chinese patent CN202210103528 discloses a paraffin section method suitable for the flower buds of crape myrtle, which is carried out according to the gradient of 50 percent ethanol (30 min) -70 percent ethanol overnight-85 percent ethanol (50 min) -95 percent ethanol (50 min) -100 percent ethanol (50 min) in the dehydration process, the preparation method of the paraffin section is simple and easy to operate, and the success rate of the section is improved, and the section effect is good.
Chinese patent CN201811595700.4 discloses a method for producing paraffin section for observing flower bud differentiation process, which comprises fixing plant sample materials, dehydrating and transparentizing, embedding through paraffin, correcting wax block section, sticking piece, dyeing and sealing, wherein the fixing plant sample materials comprise: cutting off leaves which are grown on plant rhizomes, cutting off central tissues of plant shoot tips, immersing the central tissues in a container filled with FPA fixing liquid, and placing the container in a vacuum dryer to enable a sample to sink to obtain a fixed sample. The fixing method reduces the embrittlement of the tender material after fixing.
At present, the prior art has little attention on other flower tissues, and has fewer reports on related researches on different tissue parts of the phalaenopsis, and particularly, a slice with two tissues with different soft and hardness tightly connected has no related research slice making method. The process of axillary bud development and pedicel formation of commercial popular orchid with higher economic value of butterfly orchid is the hot spot and key point of the research of the orchid industry nowadays, and the quality of paraffin section production seriously influences the microscopic morphological development process observation requirement. The observation shows that the stem has serious lignification phenomenon and the flower bud tissue is very tender, so that when paraffin slicing is carried out, the stem section is tightly connected with the axillary bud in the softening stage of the test material, however, the softening time required by the lignified stem section is longer by adopting a conventional softening method due to the extremely large difference of the hardness of the stem section and the axillary bud, and the tender axillary bud is crushed due to overlong softening time; or the tender axillary buds are suitable in softening time, and the lignified stem segments are still too hard to meet the requirement of embedding in the next step, so that for treating plant tissues with two different soft hardness which are tightly connected and indistinguishable on the same plant, a conventional softening method is adopted to obtain a complete wax belt, and the conditions of flaking and lamination are often accompanied in the subsequent slicing process, so that microscopic morphological observation required by research cannot be performed.
According to the method, the operation steps of paraffin slicing are optimized according to the growth characteristics of the butterfly orchid stem and the latent bud and the difference of tissue hardness, so that young axillary buds are not damaged, and a softening effect of a harder stem is achieved well. The method for preparing the paraffin sections of the plant tissue materials with different hardness, which are required in the process of the paraffin sections of the butterfly orchid, can be applied to the preparation method of the paraffin sections of the plant tissue materials with different hardness, and can well obtain the paraffin sections with high quality for subsequent microscopic morphological observation and research.
Disclosure of Invention
The invention aims to provide a preparation method of paraffin sections of soft and hard plant tissues, which solves the problem that in the prior art, two plant tissues with different soft and hard hardness which are indistinct and tightly connected are treated by adopting a conventional paraffin section preparation method by adjusting the preparation method of the paraffin sections, so that a complete paraffin belt cannot be obtained.
In order to achieve the above object, the present invention has the following technical scheme:
in one aspect, the invention provides a method for preparing paraffin sections of soft and hard phase plant tissues, which comprises the following steps:
(1) Fixing: fixing plant tissue in a fixing solution, and pumping air for 1-3 times for 48-96 hours;
(2) Softening: adding 8-14% hydrofluoric acid softening solution, vacuumizing for 20-50 min, and softening for 10-15 days;
(3) Dehydrating: placing the softened tissue in 30% alcohol for 2 hours, 50% alcohol for 2 hours, 70% alcohol for 2 hours, 80% alcohol for 1 hour, 90% alcohol for 1 hour, absolute alcohol for 2 hours, alcohol benzene for 1 hour, xylene for 2 hours;
(4) Wax dipping: immersing the dehydrated material into melted paraffin in an incubator;
(5) Embedding: embedding the waxed tissue in an embedding machine;
(6) Slicing and expanding: slicing with paraffin slicer, floating on 40deg.C warm water to flatten tissue, and baking at 40deg.C;
(7) Dewaxing and rehydration: placing the slice in xylene for 8 minutes-3 seconds in pure xylene and absolute ethanol-3 seconds in 90% alcohol-3 seconds in 80% alcohol-3 seconds in 70% alcohol-3 seconds in 50% alcohol-3 seconds in 30% alcohol, 3 seconds in water;
(8) Dyeing: placing the plant tissue slice into safranin dye liquor, washing with water, keeping in 50% alcohol for 3 seconds, keeping in 70% alcohol for 3 seconds, airing with equal alcohol, placing the slice into solid green dye liquor, and rapidly dehydrating the slice with absolute alcohol for three times for 3 seconds each time;
(9) Sealing piece: cut into xylenes and sealed with neutral resin.
Preferably, the plant tissue in step (1) is a phalaenopsis plant tissue.
Further preferably, the plant tissue is taken from the part of the pedicel that is connected to the stem bud.
Further preferably, the plant tissue has a size of 3-10 mm diameter and a length of 12-20mm.
Preferably, the fixative solution in step (1) is a FAA fixative solution.
Further preferably, the FAA fixative solution comprises 5% formalin, 5% glacial acetic acid and 90% ethanol at a mass fraction of 50%.
Further preferably, the mass-to-volume ratio of the plant tissue to the fixative solution is 1:20.
Preferably, the number of times of air suction in the step (1) is 3, and the fixed time is 48 hours.
Preferably, the concentration of hydrofluoric acid in step (2) is 10%.
Preferably, the softening time in step (2) is 12 days.
Preferably, the mass-to-volume ratio of the plant tissue to the softening liquid in the step (2) is 1:20.
Preferably, the time for the evacuation in step (2) is 30 minutes.
Preferably, after evacuation, it is placed in a blow-drying oven at 55℃for softening.
Preferably, after softening, it is placed in running water overnight and washed to neutrality.
Preferably, the temperature of the incubator in step (4) is 65 ℃.
Preferably, the step (4) of wax dipping is specifically that after the dehydration is completed, the material is immersed in melted paraffin for 1h in a constant temperature box at 65 ℃, the melted paraffin is replaced, immersed in melted paraffin for 1d, the melted paraffin is replaced again, and immersed in melted paraffin for 1d.
Preferably, after the embedding in step (5) is completed and the wax is solidified, the modified wax block is removed from the embedding frame.
Preferably, the thickness of the slice in step (6) is 4 μm.
Preferably, the volume ratio of the pure xylene to the absolute ethanol in the mixed solution of the pure xylene and the absolute ethanol in the step (7) is 1:1.
Preferably, the plant tissue sections are maintained in the safranin dye solution in step (8) for a period of time of 1-2 minutes.
Preferably, the time for slicing into solid green dye solution in step (8) is 1-5 minutes.
Preferably, the slices in step (9) are transparent to xylene for 5 minutes.
Specifically, the lignified cells in the main stem of the butterfly orchid of the present invention are blue-green, and the other cells are red.
In yet another aspect, the invention provides paraffin sections prepared by the above preparation method.
Specifically, the paraffin section is a butterfly orchid paraffin section.
In yet another aspect, the invention provides a paraffin section prepared by the preparation method or the application of the paraffin section in plant tissue organ or cell observation.
Preferably, the plant tissue is butterfly orchid plant tissue.
The beneficial effects of the invention are as follows:
the method optimizes the preparation steps of paraffin sections, wherein the method comprises the steps of softening liquid, softening time, softening step, dehydration and the like, and the vacuum treatment during softening can help the softening agent to quickly infiltrate into a sample, so that the softening efficiency is effectively improved.
Drawings
FIG. 1 is a plant tissue material diagram of example 1.
Fig. 2 is a display sheet of example 1.
FIG. 3 is a microscopic view of paraffin sections of example 1.
FIG. 4 is a microscopic view of paraffin sections of example 2.
FIG. 5 is a microscopic view of paraffin sections of example 3.
FIG. 6 is a microscopic view of paraffin sections of example 4.
FIG. 7 is a microscopic view of paraffin sections of example 5.
FIG. 8 is a graph of plant tissue material of comparative example 1.
Fig. 9 is a display of comparative example 1.
FIG. 10 is a microscopic view of paraffin sections of comparative example 2.
FIG. 11 is a microscopic view of paraffin sections of comparative example 3.
FIG. 12 is a microscopic view of paraffin sections of comparative example 4.
Fig. 13 is a microscopic view of paraffin section of comparative example 5.
Fig. 14 is a display of comparative example 6.
Detailed Description
In order to make the technical means, the creation features, the achievement of the purpose and the effect of the present invention easy to understand, the present invention will be further elucidated with reference to the specific embodiments, but the following embodiments are only preferred embodiments of the present invention, not all of them. Based on the examples in the embodiments, those skilled in the art can obtain other examples without making any inventive effort, which fall within the scope of the invention. In the following examples, unless otherwise specified, the methods of operation used were conventional, the equipment used was conventional, and the materials used in the examples were the same.
Experimental materials purchase manufacturer and number:
50% faa fixative: available from marchane seville biotechnology, inc., product number: g1108-500ML.
Safranin dye liquor: available from the wuhansai biotechnology company, inc. under the designation G1031-1.
Solid green dye liquor: available from the company of wuhansai biotechnology, inc. under the designation G1031-2.
EXAMPLE 1 preparation of Paraffin sections
1. Fixing
Fresh butterfly orchid tissue with the length of about 12 mm and the diameter of about 4 mm is taken and added into 30 mL of 50% FAA fixing solution for fixing, the mass-volume ratio of plant tissue to fixing solution is 1:20, and the fixing time is 48 hours. Meanwhile, in order to prevent the material from being heated to release air in the later wax block embedding process, bubbles appear on the surface to influence the quality of the slice, the fixing liquid is pumped for 3 times until no bubbles are generated, and the air in the cell gap in the material can be effectively removed.
2. Softening of
Placing the embedding frame into a plastic dyeing jar, pouring 30 mL 10% hydrofluoric acid softening solution, vacuumizing for 30 minutes, sealing, and placing into a 55 ℃ blast drying box for softening for 12 days. After softening, putting the fabric into flowing water for overnight washing to be neutral, and facilitating the subsequent successful dyeing.
3. Dewatering
Placing the dehydration box into a basket, and sequentially carrying out gradient alcohol dehydration in a dehydrator:
(1) Hold in 30% alcohol for 2 hours;
(2) Hold in 50% alcohol for 2 hours;
(3) Hold in 70% alcohol for 2 hours;
(4) Hold in 80% alcohol for 1 hour;
(5) Hold in 90% alcohol for 1 hour;
(6) Holding in absolute ethanol for 2 hours;
(7) Hold in alcohol benzene for 1 hour;
(8) The reaction mixture was kept in xylene for 2 hours.
4. Wax dipping
After dehydration, the material is immersed in a constant temperature box at 65 ℃ for 1h, the melted paraffin is replaced, immersed in the melted paraffin for 1d, replaced again, and immersed in the melted paraffin for 1d.
5. Embedding
Embedding the wax-soaked tissue in an embedding machine. Firstly, putting melted wax into an embedding frame, taking out tissues from a dehydration box before the wax is solidified, putting the tissues into the embedding frame according to the requirement of an embedding surface, and attaching corresponding labels. Cooling at-20deg.C, solidifying, removing the wax block from the embedding frame, and trimming.
6. Slicing and expanding sheet
The trimmed wax block was placed in a paraffin microtome for slicing, and the thickness was selected to be 4. Mu.m. The slices float on warm water at 40 ℃ of a slice spreading machine to flatten the tissues, the glass slide drags the tissues out, superfluous distilled water is removed, and the slices are baked in an oven at 40 ℃. And (5) baking the water, drying the wax, baking, taking out and preserving at normal temperature for standby.
7. Dewaxing and rehydration
(1) The sections were placed in xylene for 8 minutes;
(2) Maintaining in the mixed solution of pure xylene and absolute ethanol (volume ratio 1:1) for 3 seconds;
(3) Holding in absolute ethanol for 3 seconds;
(4) Hold in 90% alcohol for 3 seconds;
(5) Hold in 80% alcohol for 3 seconds;
(6) Hold in 70% alcohol for 3 seconds;
(7) Hold in 50% alcohol for 3 seconds;
(8) Hold in 30% alcohol for 3 seconds;
(9) The mixture was kept in pure water for 3 seconds.
8. Safranine fast green dyeing
(1) Placing the plant tissue slices into safranin dye liquor for 1-2 minutes, and washing with water;
(2) Hold in 50% alcohol for 3 seconds;
(3) Hold in 70% alcohol for 3 seconds;
(4) After the alcohol is dried, slicing the mixture into solid green dye liquor for 1 minute;
(5) The slices were rapidly dehydrated with three times of absolute ethanol for 3s each time.
9. Transparent sealing sheet
The sections were transparent to clean xylene for 5 minutes and sealed with neutral resin. Cover glass is covered by slowly moving from the side, and the cover glass is dried in an incubator at 40 ℃ or is placed in a room for natural airing and labeling. The invention can obviously distinguish the lignified cells in the main stem of the butterfly orchid from other cells in red, and the invention can spread out the film for shooting.
Experimental results:
the experimental results are shown in fig. 1-3, and it can be seen from fig. 1-3 that the paraffin section prepared in this embodiment has better quality and higher integrity, and the cells of the tissue materials with two soft and hard connected after treatment are relatively intact, which indicates that the optimized paraffin section preparation method of the invention can better maintain the integrity of the whole cells of the tissue of the material in the section, clearly distinguish soft tissue from lignified harder tissue, and simultaneously develop good microscopic morphology observation on two closely connected different tissue materials.
EXAMPLE 2 preparation of Paraffin sections
Example 2 differs from example 1 in that the softening step (2) is different, the rest being the same, the softening operation is as follows:
placing the embedding frame into a plastic dyeing jar, pouring 10% hydrofluoric acid softening solution, vacuumizing for 30 minutes, sealing, and placing into a 55 ℃ blast drying oven for softening for 15 days. After softening, the mixture was put into running water overnight and washed to neutrality.
The experimental results are shown in fig. 4, and paraffin sections are very effective.
EXAMPLE 3 preparation of Paraffin sections
Example 3 differs from example 1 in that the softening step (2) is different, the rest being the same, the softening step being as follows:
placing the embedding frame into a plastic dyeing jar, pouring 12% hydrofluoric acid softening solution, vacuumizing for 30 minutes, sealing, and placing into a 55 ℃ blast drying oven for softening for 12 days. After softening, the mixture was put into running water overnight and washed to neutrality.
The experimental results are shown in fig. 5, and paraffin sections are very effective.
EXAMPLE 4 preparation of Paraffin sections
Example 4 differs from example 1 in that the softening step (2) is different, the rest being the same, the softening step being as follows:
placing the embedding frame into a plastic dyeing jar, pouring 12% hydrofluoric acid softening solution, vacuumizing for 30 minutes, sealing, and placing into a 55 ℃ blast drying oven for softening for 15 days. After softening, the mixture was put into running water overnight and washed to neutrality.
The experimental results are shown in FIG. 6, and paraffin sections are very effective.
EXAMPLE 5 preparation of Paraffin sections
Example 5 differs from example 1 in that the time of vacuum operation during softening is different, and the time of vacuum operation in example 5 is 50 minutes, and the rest is the same.
The experimental results are shown in fig. 7, and the vacuumizing time is 50 minutes, and the effect is not obviously different from that of vacuumizing for 30 minutes.
Comparative example 1 preparation of paraffin sections
The conventional paraffin section method was used in comparative example 1, and was different from example 1 in the steps of fixing and softening, which were the same as the rest, specifically comprising the steps of:
1. fixing
And cleaning the main stem, fixing fresh tissues in 50% FAA fixing solution for 24 hours, taking out the tissues from the fixing solution, trimming the tissues of the target part in a fume hood by using a surgical knife, and cleaning the trimmed tissues and the corresponding labels in a dehydration disc.
2. Softening of
The embedding frame is placed into a plastic staining jar, and is poured into a mixed solution (volume ratio is 1:1) of 50% alcohol and 70% glycerol to soften for 15 days.
Experimental results:
the experimental results are shown in fig. 8-9, and it can be seen from fig. 8-9 that the conventional treatment method has poor slicing effect, the wax belt is crushed during slicing, and the middle sample tissue is obviously damaged.
Comparative example 2 preparation of paraffin sections
Comparative example 2 is different from example 1 in that the concentration of hydrofluoric acid is different during the paraffin section preparation process, and the concentration of hydrofluoric acid in comparative example 2 is 5%, and the rest is the same.
As a result of the experiment, as shown in fig. 10, it can be seen from fig. 10 that when the concentration of hydrofluoric acid is 5%, the softening effect of comparative example 2 is poor and the dyeing effect is affected, also as in the case of the treatment of example 1 for 12 days.
Comparative example 3 preparation of paraffin sections
Comparative example 3 is different from example 1 in that the concentration of hydrofluoric acid is different during the paraffin section preparation process, and the concentration of hydrofluoric acid in comparative example 3 is 15%, and the rest is the same.
As shown in fig. 11, when the concentration of hydrofluoric acid is high, the experimental result is shown in fig. 11, and it is obvious that a void appears in the middle of the material, and the embedding and slicing effects are poor.
Comparative example 4 preparation of paraffin sections
Comparative example 4 is different from example 1 in that the concentration of hydrofluoric acid and the treatment time are different in the paraffin section preparation process, the concentration of hydrofluoric acid in comparative example 4 is 15%, the treatment time is 15 days, and the rest is the same.
As shown in fig. 12, the experimental result shows that when the concentration of hydrofluoric acid is 15%, the treatment time is 15 days, more holes are formed in the middle of the material, and the embedding slicing effect is poorer.
Comparative example 5 preparation of paraffin sections
Comparative example 5 differs from example 1 in that the paraffin section was softened during preparation without vacuum operation, and the remainder were the same.
The experimental results are shown in fig. 13, and the subsequent dyeing is affected without vacuumizing operation in the softening process, so that the effect is poor.
Comparative example 6 preparation of paraffin sections
Comparative example 6 differs from example 1 in that the dehydration step during the preparation of paraffin sections, starting with a higher concentration of 70%, is followed by the following steps:
placing the dehydration box into a basket, and sequentially carrying out gradient alcohol dehydration in a dehydrator:
(1) Hold in 70% alcohol for 2 hours;
(2) Hold in 80% alcohol for 1 hour;
(3) Hold in 90% alcohol for 1 hour;
(4) The mixture was kept in absolute ethanol for 2 hours.
The experimental results are shown in fig. 14, the dehydration is carried out from the higher concentration, so that the sample tissue is rapidly dehydrated and shrunken, the subsequent wax dipping and embedding can be affected, and the effect is poor.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Claims (10)
1. The preparation method of the soft and hard phase plant tissue paraffin section is characterized by comprising the following steps:
(1) Fixing: fixing plant tissue in a fixing solution, and pumping air for 1-3 times for 48-96 hours;
(2) Softening: adding 8-14% hydrofluoric acid softening solution, vacuumizing for 20-50 min, and softening for 10-15 days;
(3) Dehydrating: placing the softened tissue in 30% alcohol for 2 hours, 50% alcohol for 2 hours, 70% alcohol for 2 hours, 80% alcohol for 1 hour, 90% alcohol for 1 hour, absolute alcohol for 2 hours, alcohol benzene for 1 hour, xylene for 2 hours;
(4) Wax dipping: immersing melted paraffin in an incubator after dehydration is completed;
(5) Embedding: embedding the waxed tissue in an embedding machine;
(6) Slicing and expanding: slicing with paraffin slicer, floating on 40deg.C warm water to flatten tissue, and baking at 40deg.C;
(7) Dewaxing and rehydration: placing the slice in xylene for 8 minutes-3 seconds in pure xylene and absolute ethanol-3 seconds in 90% alcohol-3 seconds in 80% alcohol-3 seconds in 70% alcohol-3 seconds in 50% alcohol-3 seconds in 30% alcohol, 3 seconds in water;
(8) Dyeing: placing the plant tissue slice into safranin dye liquor, washing with water, keeping in 50% alcohol for 3 seconds, keeping in 70% alcohol for 3 seconds, airing with equal alcohol, placing the slice into solid green dye liquor, and rapidly dehydrating the slice with absolute alcohol for three times for 3 seconds each time;
(9) Sealing piece: cut into xylenes and sealed with neutral resin.
2. The method according to claim 1, wherein the concentration of hydrofluoric acid is 10%.
3. The method of claim 1, wherein the softening time is 12 days.
4. The method of claim 1, wherein the time for evacuating is 30 minutes.
5. The method according to any one of claims 1 to 4, wherein the plant tissue in the step (1) is a phalaenopsis plant tissue.
6. The method according to any one of claims 1 to 4, wherein the plant tissue is obtained from a portion where a pedicel is connected to a shoot.
7. The method according to any one of claims 1 to 4, wherein the plant tissue has a size of 3 to 10 mm in diameter and a length of 12 to 20mm.
8. Paraffin sections prepared by the preparation method according to any one of claims 1 to 7.
9. Use of paraffin sections according to claim 8 for the observation of plant tissue organs or cells.
10. The use according to claim 9, wherein said plant tissue is butterfly orchid plant tissue.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311101420.4A CN116840020B (en) | 2023-08-30 | 2023-08-30 | Preparation method of paraffin section of soft and hard phase plant tissue |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311101420.4A CN116840020B (en) | 2023-08-30 | 2023-08-30 | Preparation method of paraffin section of soft and hard phase plant tissue |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN116840020A true CN116840020A (en) | 2023-10-03 |
| CN116840020B CN116840020B (en) | 2023-11-17 |
Family
ID=88163818
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311101420.4A Active CN116840020B (en) | 2023-08-30 | 2023-08-30 | Preparation method of paraffin section of soft and hard phase plant tissue |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116840020B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108693010A (en) * | 2018-04-04 | 2018-10-23 | 南京烁朴生物科技有限公司 | A kind of plant tissue softening agent and application method |
| CN111257089A (en) * | 2020-02-18 | 2020-06-09 | 广东省农业科学院环境园艺研究所 | Optimized manufacturing method of paraffin sections of different flower tissues of butterfly orchid |
| CN115683697A (en) * | 2022-09-09 | 2023-02-03 | 安徽农业大学 | Method for slicing plant phloem tissue |
-
2023
- 2023-08-30 CN CN202311101420.4A patent/CN116840020B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108693010A (en) * | 2018-04-04 | 2018-10-23 | 南京烁朴生物科技有限公司 | A kind of plant tissue softening agent and application method |
| CN111257089A (en) * | 2020-02-18 | 2020-06-09 | 广东省农业科学院环境园艺研究所 | Optimized manufacturing method of paraffin sections of different flower tissues of butterfly orchid |
| CN115683697A (en) * | 2022-09-09 | 2023-02-03 | 安徽农业大学 | Method for slicing plant phloem tissue |
Non-Patent Citations (2)
| Title |
|---|
| 周乃富 等: "木本植物非均质化组织石蜡切片制作方法", 植物学报, vol. 53, no. 5, pages 653 - 659 * |
| 苏印泉 等: "桦树松萝的石蜡切片方法改良及形态学研究", 西北植物学报, vol. 27, no. 5, pages 859 - 863 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN116840020B (en) | 2023-11-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Smith | The acetocarmine smear technic | |
| CN102607907B (en) | Paraffin section method for fern gametophytes | |
| CN103630421A (en) | Production method for paraffin section of paeonia lactiflora mature embryo | |
| CN107702959A (en) | A kind of paraffin section method for vegetable material serial dehydration | |
| Postek et al. | A new short chemical dehydration method for light microscopy preparations of plant material | |
| JP6876193B2 (en) | How to obtain seedlings of Hyakusanso cold cedar by embryo rescue technology in a short time | |
| CN110926851B (en) | Method for obtaining vascular cambium of woody plant and application thereof | |
| CN111257089B (en) | Optimized manufacturing method of paraffin sections of different flower tissues of butterfly orchid | |
| CN109374373A (en) | A kind of Apple paraffin section fast method for preparing | |
| CN107132097B (en) | Slicing method suitable for observing anatomical structures of flower organs of rubber trees | |
| CN116840020B (en) | Preparation method of paraffin section of soft and hard phase plant tissue | |
| CN107655731B (en) | Paraffin section method for leaf buds and flower buds of larger explants of carnation | |
| CN106417033B (en) | A kind of Gaotang snakegourd rapid propagation method | |
| CN105890919A (en) | Corn primary root tissue freeze-slicing method | |
| CN108332989B (en) | Method for analyzing high-starch-content cereal grains and observing internal structure | |
| Debenham | A modified technique for the microscopic examination of the xylem of whole plants or plant organs | |
| CN108333013A (en) | A kind of preparation method of jujube flower paraffin section | |
| CN115683697A (en) | Method for slicing plant phloem tissue | |
| CN111141568A (en) | Method for obtaining phloem of fast-growing tree species and application thereof | |
| CN107976353B (en) | Preparation method of crop tender root paraffin slice | |
| CN110793829A (en) | Method for slicing highland barley stem profile paraffin suitable for plateau environment | |
| CN105136546A (en) | Paraffine slicing method for preventing maple leaf plants from leaf anthocyanin loss | |
| CN112903406B (en) | Method for making paraffin section of tree thin root | |
| CN104458387B (en) | The fast green method transparent to color separation oil blackeite during dye of sarranine | |
| CN114235536B (en) | Slice preparation method for observing lignin deposition of stems of two-year-old masson pine |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |