CN113702131A - Paraffin section of camellia oleifera flower bud and preparation method and application thereof - Google Patents
Paraffin section of camellia oleifera flower bud and preparation method and application thereof Download PDFInfo
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- CN113702131A CN113702131A CN202111038766.5A CN202111038766A CN113702131A CN 113702131 A CN113702131 A CN 113702131A CN 202111038766 A CN202111038766 A CN 202111038766A CN 113702131 A CN113702131 A CN 113702131A
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- G—PHYSICS
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- 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
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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
- 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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Abstract
The invention discloses a paraffin section of camellia oleifera flower buds as well as a preparation method and application thereof. The paraffin section prepared by the method has high quality, clear and complete tissue structure, obvious effect, strong operability, high repeatability and strong practicability; the method provided by the invention can be used for carrying out paraffin section on the camellia oleifera flower buds at different development stages so as to observe the whole camellia oleifera flower development process.
Description
Technical Field
The invention belongs to the technical field of paraffin section, particularly relates to a paraffin section and a preparation method and application thereof, and particularly relates to a paraffin section of camellia oleifera flower buds and a preparation method and application thereof.
Background
Camellia oleifera is (Camellia oleifera Abel.) of Camellia (Theaceae) and is native to China, and is called four woody oil plants in the world together with oil palm, coconut and olive. The camellia seed oil is rich in unsaturated fatty acid, vitamin E, camellin and other specific physiological active substances, has extremely high edible and medical health care values, and is deeply loved by the vast consumers.
The flowering of plants is an important basis for economic yield formation of plants, and the camellia oleifera is in the same male and female flowers, so that the yield is low due to the problems of extremely low selfing rate, serious flower and fruit dropping and the like. Through the observation of the anatomical structures of the camellia oleifera flower buds at different periods, the key period of the growth and development of the camellia oleifera flower buds can be found, and corresponding cultivation measures are taken on the basis of the key period, so that the problem that the camellia oleifera is more flowers and less fruits in the production process is solved. Meanwhile, the research on the anatomical structure of the camellia oleifera flower bud can help to identify germplasm resources, so that the method for effectively obtaining the camellia oleifera flower bud slices has important application value in identifying and evaluating a large amount of camellia oleifera germplasm, and is beneficial to shortening the identification period of the camellia oleifera germplasm resources.
Paraffin section is one of important test techniques in histology, is used for observing morphological structures of cells and tissues, and is widely applied to research in various subject fields. The paraffin section basic steps comprise a series of treatments such as sampling and fixing, softening, dehydrating, transparentizing, waxing, embedding, wax repairing, slicing, spreading, drying, dewaxing, rehydration, dyeing, sealing, observing and the like, the preparation steps are complex, the period is long, each step is important, and the treatment methods required by plants with different tissue structures and tissues of different parts of the plants are different. The slicing quality is affected by problems in the processing method or operation, and the problems of material breakage, incomplete slicing, discontinuous wax band, existence of cavities and the like are frequently caused.
The camellia oleifera flower bud is dense in tissue structure, has difficulty in links such as dehydration, wax dipping and slicing, is easy to cause the problems of incomplete slicing, unclear tissue structure and the like due to improper treatment, and is not beneficial to observation. At present, the research on the paraffin slicing technology by taking the camellia oleifera flower buds as the materials is less, the slicing effect is not ideal enough, and the camellia oleifera flower bud slices with more complete and clear structures are not reported.
Disclosure of Invention
The paraffin section prepared by the method has high quality, clear and complete tissue structure, obvious effect, strong operability, high repeatability and strong practicability; the method provided by the invention can be used for carrying out paraffin section on the camellia oleifera flower buds at different development stages so as to observe the whole camellia oleifera flower development process.
Therefore, the technical scheme of the invention is as follows:
in a first aspect, the invention provides a method for making paraffin sections of camellia oleifera flower buds, which at least comprises the following steps:
(1) material taking, dividing and fixing: picking up camellia oleifera flower buds, stripping sepals, cutting off part of tissue of the camellia oleifera flower buds, fixing and softening;
(2) first dehydration and first transparency treatment: dehydrating the softened camellia oleifera flower buds by adopting gradient alcohol in sequence; then carrying out a clearing treatment, wherein the clearing agent at least comprises n-butanol;
the first dehydration mode is that ethanol water solution with volume concentration of 70%, 85% and 95% and absolute ethanol are adopted for soaking in sequence, the absolute ethanol is replaced for soaking, and the soaking time is at least 1 h;
(3) wax dipping: immersing the transparent camellia oleifera flower buds into an analytically pure n-butanol-paraffin mixed solution and a pure paraffin solution in sequence;
(4) embedding, wax trimming, slicing, sticking, spreading and baking the waxed camellia oleifera flower buds in sequence; wherein the thickness of the camellia oleifera flower bud slice wax band is preferably 10-15 μm; the sticky tablet is prepared by mixing fresh egg white and analytically pure glycerin according to the volume ratio of 1: 1, configuring; the slide spreading time is at least 30min, and the slide drying time is at least 12 h;
(5) then carrying out dewaxing, rehydration and dyeing treatment; wherein, the dewaxing method comprises the specific steps of immersing the glass fiber in a xylene solution for dewaxing for 20min, and then replacing the xylene solution for dewaxing for 20 min; soaking the dewaxed camellia oleifera flower bud slices in a rehydration agent for rehydration for 10min to obtain pre-rehydrated camellia oleifera flower bud slices; the rehydration agent is analytically pure dimethylbenzene and absolute ethyl alcohol according to a volume ratio of 1: 1, mixing; soaking the pre-rehydrated camellia oleifera flower bud slices in absolute ethyl alcohol and ethanol aqueous solutions with the volume concentrations of 95%, 85% and 70% of the absolute ethyl alcohol in sequence for rehydration to obtain rehydrated camellia oleifera flower bud slices; soaking in a safranine coloring agent, sequentially soaking in 85% and 95% ethanol aqueous solution, and soaking with a green-fixing coloring agent to obtain a stained camellia oleifera flower bud slice; the safranin staining agent is a 1% safranin solution, the solute is safranin or safranin powder solvent is an ethanol water solution with the volume concentration of 50%, the fast green staining agent is a 1% fast green solution, and the solute is a fast green powder solvent is an ethanol water solution with the volume concentration of 95%;
(6) second dehydration and second transparency treatment: dehydrating the dyed camellia oleifera flower buds by adopting gradient alcohol in sequence; then carrying out transparent treatment, wherein the transparent agent is dimethylbenzene;
(7) sealing: and sealing the slices with neutral resin, and drying to obtain paraffin sections of the camellia oleifera flower buds.
Preferably, in the step (1), the tissue of the camellia oleifera flower bud 1/3-1/5 volume is cut off longitudinally; above-mentioned processing is favorable to the fixed of bud tissue, operations such as dehydration, waxing for inside solution can immerse bud tissue, adjustment position when being favorable to the embedding simultaneously, the tangent plane is placed the level down and is made the bud be on a parallel with embedding box bottom surface all the time when embedding, can cut the bud central authorities fast and contain the complete longitudinal section of structures such as stamen pistil during the section, and then conveniently observe the development change of bud internal cell and organizational structure. Because the camellia oleifera flower bud has a compact structure, repeated vacuum pumping treatment during fixation and softening is beneficial to fully immersing the solution into camellia oleifera flower bud tissues for fixation and softening cells and tissues, so that the change of morphological structures such as atrophy and the like caused by cell decay can be prevented, and the integrity of the cells is further maintained;
preferably, the length of the exposed stamen after incision is taken as a standard:
for camellia oleifera flower buds smaller than 1.1cm (for example, 1.0cm, 0.9cm, 0.8cm, 0.7cm, 0.6cm, 0.5cm and the like or all values in the range, which are not described in detail due to space limitation), tissues with the volume of the camellia oleifera flower buds 1/5 are longitudinally cut;
for camellia oleifera floral buds greater than 1.1cm (e.g., 1.1cm, 1.2cm, 1.3cm, 1.4cm, 1.5cm, 1.6cm, 1.7cm, 1.8cm, 1.9cm, 2.0cm, etc., or all values within the stated range, which will not be described again due to space limitations), the tissue of volume of camellia oleifera floral bud 1/3 is cut longitudinally;
the cutting is not performed for camellia oleifera flower buds with length less than 0.5cm (for example, 0.4cm, 0.3cm, 0.2cm, 0.1cm, and the like or all values in the range, which are not described in detail due to space limitation).
Wherein, the fixing solution preferably comprises analytically pure formaldehyde solution, glacial acetic acid and ethanol water solution with volume concentration of 70% according to a volume ratio of 1: 1: 18 configuration; the softening liquid is prepared by mixing 70% ethanol aqueous solution and analytically pure glycerin according to the volume ratio of 1: 1 configuration.
Preferably, the transparent treatment in the step (2) is specifically:
the dehydrated camellia oleifera flower buds are soaked in the first transparent agent and the second transparent agent in sequence and are replaced for at least 3 times, and the volume ratio of the dehydrated camellia oleifera flower buds to the solution of the transparent agents is more than 1: 10 (e.g., 1: 10, 1: 9.5, 1: 9, 1: 8.5, 1: 8, 1: 7.5, 1: 7, 1: 6.5, 1: 6, 1: 5.5, 1: 5 or all values within the ranges, which are not described herein again due to space limitations);
wherein, the first clearing agent is analytically pure n-butyl alcohol and analytically pure ethanol according to the volume ratio of 1: 1, mixing the solution;
and the second transparent agent is analytically pure butanol. The n-butyl alcohol is used as the transparent agent for transparent treatment, and is different from the traditional method of using dimethylbenzene as the transparent agent, the toxicity of the n-butyl alcohol is far lower than that of the dimethylbenzene, the n-butyl alcohol is good in transparent effect after being used as the transparent agent, the tissue is flat, the phenomena of obvious shrinkage and deformation do not exist, the problem that the material becomes brittle due to overlong transparent operation time does not exist, the slicing result is ensured, and meanwhile, the harm of a dimethylbenzene solution to experimenters is reduced to a great extent;
preferably, the analytically pure butanol-paraffin mixed liquor obtained in the step (3) is completely melted and mixed in advance in a high-temperature environment, and is different from a traditional wax dipping method of gradually adding broken wax, the time and the labor are saved when the analytically pure butanol-paraffin mixed liquor is melted and mixed in advance, and the wax dipping effect is good.
Preferably, the temperature of the high temperature environment is 75-85 ℃, for example, 75 ℃, 76 ℃, 77 ℃, 78 ℃, 79 ℃, 80 ℃, 81 ℃, 82 ℃, 83 ℃, 84 ℃, 85 ℃ and all values in the range can be used, and the description is omitted for reasons of space limitation;
preferably, the mass-to-volume ratio of the analytically pure butanol to the paraffin is 1: 1 g/mL;
preferably, the step (3) of soaking further comprises the following steps:
baking at 55-65 deg.C, and soaking in liquid paraffin to obtain wax-soaked camellia oleifera flower bud; the replacement is carried out for at least 3 times, the first soaking time and the second soaking time are 4 hours, the third soaking time is 12 hours, the melting point of the liquid paraffin is 58-60 ℃, and the volume ratio of the camellia oleifera flower bud material to the paraffin is 1: 50;
preferably, the temperature of the liquid paraffin is 55-65 ℃, for example, 55 ℃, 56 ℃, 57 ℃, 58 ℃, 59 ℃, 60 ℃, 61 ℃, 62 ℃, 63 ℃, 64 ℃, 65 ℃ and all values in the range can be used, and the description is omitted for brevity. The temperature of the baking and liquid paraffin should not exceed 65 ℃, too high a temperature may cause carbonization of the sample, and too low a temperature may not achieve the purpose of melting paraffin.
Preferably, the transparent treatment in the step (6) is specifically:
soaking the dehydrated and dyed camellia oleifera flower buds by using a third clearing agent and a fourth clearing agent in sequence;
wherein, the third clearing agent is analytically pure dimethylbenzene and analytically pure ethanol according to the volume ratio of 1: 1, mixing the solution;
the clearing agent IV is analytically pure dimethylbenzene; the soaking time is at least 5min each time;
the second dehydration method comprises sequentially soaking in 95% ethanol solution and anhydrous ethanol for at least 5 min.
Preferably, the steps requiring heating and constant temperature treatment, such as softening, waxing, sheet spreading, sheet baking and the like, are all performed in the oven, and the method can replace the functions of a sheet spreading machine and a sheet baking machine, effectively save the cost required by operation, and simultaneously can manufacture high-quality slices.
As a preferred technical solution, the manufacturing method at least comprises the following steps:
(1) material taking, dividing and fixing: picking up camellia oleifera flower buds, stripping sepals, cutting off part of tissue of the camellia oleifera flower buds, fixing and softening; the length of the exposed stamen after cutting is taken as a standard:
for camellia oleifera flower buds smaller than 1.1cm, tissues with the volume of 1/5 camellia oleifera flower buds are longitudinally cut;
for camellia oleifera flower buds larger than 1.1cm, tissues with the volume of 1/3 camellia oleifera flower buds are longitudinally cut;
cutting camellia oleifera flower buds with the length of less than 0.5 cm;
(2) first dehydration and first transparency treatment: dehydrating the softened camellia oleifera flower buds by adopting gradient alcohol in sequence; the dehydrated camellia oleifera flower buds are soaked in the first transparent agent and the second transparent agent in sequence and are replaced for at least 3 times, and the volume ratio of the dehydrated camellia oleifera flower buds to the solution of the transparent agents is more than 1: 10;
wherein, the first clearing agent is analytically pure n-butyl alcohol and analytically pure ethanol according to the volume ratio of 1: 1, mixing the solution;
the second transparent agent is analytically pure butanol;
(3) wax dipping: soaking transparent camellia oleifera flower buds in an analytically pure butanol paraffin mixed solution; the analytically pure butanol paraffin mixed liquor is completely melted and mixed in advance in an environment of 75-85 ℃; the mass-volume ratio of the pure butanol to the paraffin is 1: 1 g/mL; baking at 55-65 deg.C, and soaking in 55-65 deg.C liquid paraffin to obtain wax-soaked camellia flower bud;
(4) embedding, wax trimming, slicing, sticking, spreading and baking the waxed camellia oleifera flower buds in sequence;
(5) then carrying out dewaxing, rehydration and dyeing treatment;
(6) second dehydration and second transparency treatment: dehydrating the dyed camellia oleifera flower buds by adopting gradient alcohol in sequence; soaking the dehydrated and dyed camellia oleifera flower buds by using a third clearing agent and a fourth clearing agent in sequence;
wherein, the third clearing agent is analytically pure dimethylbenzene and analytically pure ethanol according to the volume ratio of 1: 1, mixing the solution;
the clearing agent IV is analytically pure dimethylbenzene;
(7) sealing: and sealing the slices with neutral resin, and drying to obtain paraffin sections of the camellia oleifera flower buds.
In a second aspect, the present invention provides a paraffin section of camellia oleifera flower buds, which is prepared by the method of the first aspect of the present invention.
In a third aspect, the invention provides the application of the camellia oleifera flower bud paraffin section as described in the first aspect in observing tissues, organs and cells, researching related pathology of camellia oleifera flower bud tissues and breeding camellia oleifera varieties.
Compared with the prior art, the paraffin section of the camellia oleifera flower bud and the preparation method thereof provided by the invention have the advantages that the cutting treatment before fixation of the camellia oleifera flower bud is beneficial to the operations of fixation, dehydration, wax immersion and the like of the flower bud tissue, so that the solution can be immersed into the flower bud tissue to fix and soften cells, the change of the morphological structure such as atrophy and the like caused by cell decay can be prevented, the integrity of the cells is further kept, and the rapid and complete fixation of the morphological structure of the camellia oleifera flower bud is facilitated; simultaneously the position is adjusted when still being favorable to the embedding, and the tangent plane is placed to the level down and is made the flower bud be on a parallel with the embedding box bottom surface during the embedding all the time, can cut the flower bud central authorities fast and contain the complete longitudinal section of structure such as stamen pistil during the section, and then conveniently observe the development change of the inside cell of flower bud and organizational structure. Next, n-butanol was used as a clearing agent in the clearing treatment. Because the toxicity of xylene in the traditional clearing agent is high, and the toxicity of n-butyl alcohol is far lower than that of xylene, the clearing agent has good clearing effect after being used as the clearing agent, smooth tissue, no obvious contraction and deformation phenomena, and the problem that the material becomes brittle due to overlong clearing operation time does not exist, so that the harm of xylene solution to experimenters is reduced to a great extent while the slicing result is ensured. And thirdly, the n-butyl alcohol paraffin melting mixed solution which is melted and mixed completely in advance is used as the wax dipping solution, the treatment is different from the traditional wax dipping method of gradually adding broken wax, the melting and mixing in advance are time-saving and labor-saving, and the wax dipping effect is good.
Drawings
FIG. 1 is a microscopic image of a paraffin section prepared in example 1,
FIG. 1a is a section of camellia oleifera flower bud under 0.63-fold microscope,
FIG. 1b is a partial enlarged view (10 times) of the camellia oleifera flower bud slice of example 1,
FIG. 1c is an enlarged view (40-fold) of the anther tissue of the camellia oleifera floral bud slice in example 1;
FIG. 2 is a microscopic image of a paraffin section prepared in example 2,
FIG. 2a is a section of camellia oleifera flower bud under 0.63-fold microscope,
FIG. 2b is a partial enlarged view (10 times) of the camellia oleifera flower bud slice of example 2,
FIG. 2c is an enlarged view (40 times) of the anther tissue of the camellia oleifera floral bud slice in example 2;
FIG. 3 is a microscopic view of a paraffin section prepared in comparative example 1,
FIG. 3a is a section of camellia oleifera flower bud under 0.63-fold microscope,
FIG. 3b is a partial enlarged view (10 times) of the camellia oleifera flower bud slice of comparative example 1,
FIG. 3c is an enlarged view (40-fold) of the anther tissue of the camellia oleifera floral bud slice of comparative example 1.
Detailed Description
The present invention will be further described with reference to the following examples, which are not intended to limit the invention in any way. The following examples and comparative examples tested materials were collected from the flower buds of camellia oleifera at different developmental stages from 8 months to 12 months 2020, planted in the "three nursery and one garden" demonstration base of tropical camellia oleifera at the university of Hainan, the camellia germplasm repository (located in Hainan province, delirium, Nada town). The camellia oleifera plants related in the examples and the comparative examples are robust in growth and in a mature stage, have no obvious plant diseases and insect pests, and have normal flower bud development.
Example 1
The specific steps of the embodiment are as follows:
(1) material taking, dividing and fixing: using scissors or a blade and a flower handle to collect camellia oleifera flower buds together, immediately taking the camellia oleifera flower buds back to a laboratory for selection and segmentation, using a brand-new clean blade to longitudinally cut off the tissues with the volumes of the flower buds 1/3-1/5 after the sepals are stripped by using tweezers, then quickly putting the cut-off buds into a 50ml centrifugal tube filled with a stationary liquid for soaking, placing 3-5 flower buds in each tube, tightly covering a cover, marking date, processing, samples, collecting people and other information, wherein the volume ratio of the material to the stationary liquid needs to be more than 1: 20g/ml, performing vacuum pumping treatment every day, preserving at 4 ℃ for the rest of time, and fixing for 7 days to obtain fixed camellia oleifera flower buds; the length of the camellia oleifera flower bud is 1.05 cm; the fixing solution is prepared by mixing analytically pure formaldehyde solution, glacial acetic acid and 70% ethanol aqueous solution according to the volume ratio of 1: 1: 18 configuration; the vacuum treatment is to place the container filled with the fixing liquid and the material into a vacuum drier and vacuumize for 30 minutes;
(2) softening: taking the fixed camellia oleifera flower buds obtained in the step (1) out of the fixed liquid, putting the fixed camellia oleifera flower buds into a 50ml centrifugal tube filled with softening liquid, placing 3-5 flower buds in each tube, tightly covering a cover, marking information such as date, treatment, samples and people, wherein the volume ratio of the materials to the softening liquid needs to be more than 1: 20g/ml, opening the cover of the centrifugal tube, vacuumizing for 30min, and softening at 60 ℃ for 24h to obtain softened camellia oleifera flower buds; the softening liquid is prepared by mixing 70% ethanol aqueous solution and analytically pure glycerin according to the volume ratio of 1: 1, configuring;
(3) first dehydration: taking the softened camellia oleifera flower buds obtained in the step (2) out of the softening liquid, respectively filling the softened camellia oleifera flower buds into 2ml centrifuge tubes which are provided with small holes and can freely pass in and out of the solution, filling 1 flower bud in each tube, writing a number on a centrifuge tube cover by using a pencil, filling the centrifuge tubes into a centrifuge tube box with 50 holes and 2ml, opening the centrifuge tube cover, rinsing the centrifuge tube cover for 3 times by using an ethanol aqueous solution with the volume concentration of 70%, and sequentially adding 500ml of ethanol with the volume concentration of 70%, 85% of ethanol and 95% of ethanol for gradient dehydration, wherein the gradient interval is 2 h. Then, removing 95% ethanol, adding equal volume of absolute ethanol for soaking treatment for 2h, replacing absolute ethanol, and soaking for 2h to obtain dehydrated camellia oleifera flower buds;
(4) first transparency: preparing a first transparent agent in advance, pouring the absolute ethyl alcohol in the centrifugal tube box in the step (3) out, pouring 500ml of the first transparent agent for soaking for 1 hour, pouring the first transparent agent out, adding an equal volume of a second transparent agent for soaking for 2 hours, replacing the second transparent agent for soaking for 2 hours, and obtaining transparent camellia oleifera flower buds; the clearing agent is two solutions, wherein the clearing agent I is analytically pure n-butyl alcohol and analytically pure ethanol, and the volume ratio of the clearing agent I to the analytically pure ethanol is 1: 1, preparing a second transparent agent which is an analytically pure butanol solution; the first and second transparent agents can be repeatedly used for 2-3 times, and the mask and gloves can be worn with strong smell;
(5) wax dipping: filling 250g of sliced paraffin with a melting point of 58-60 ℃ and 250ml of analytically pure butanol into an empty 50-hole centrifugal tube box, putting the box into a 75 ℃ oven, baking, melting and mixing completely, putting the centrifugal tube with the sample obtained in the step (4) into the box, adjusting the oven to 65 ℃, baking for 48 hours, pouring the mixed solution, adding isovolumic pure paraffin liquid, baking and waxing for 4 hours at 65 ℃, replacing the isovolumic pure paraffin liquid every 4 hours, repeating for 3 times, and performing third waxing for 12 hours to obtain waxed camellia flower buds;
(6) embedding: using harder calendar paper to manufacture detachable embedding boxes with the length, width and height of 10cm, 2cm and 2cm respectively and fixing plates with the length, width and length of 10 cm; making a label strip with the length and the width of 2cm and 1cm by using A4 paper, and writing sample codes on the front and back surfaces of the label; preparing two tweezers and a small cup for waxing; opening an embedding machine (model: KD-BM) in advance, setting the temperature to 65 ℃, putting the new wax block into a wax cylinder for melting, and putting the wax liquid obtained in the step (5) and a centrifugal tube into a sample box of the embedding machine; during embedding, preparing a paper box light-viscous wax liquid for embedding to shape the box, and dipping a small amount of wax liquid at the bottom of the box to fix the box on a fixed plate; heating tweezers at 65 ℃, putting the prepared label into a box (right in front of the expected sample position) according to the sequence of embedding materials, then filling wax liquid into a wax cylinder by using a small cup, pouring the wax liquid into the box, slightly filling the box, then horizontally putting flower buds in the sample box by using the heated tweezers into the center of the small box according to the corresponding label position for 2-3 seconds, slightly fixing the flower buds, and timely replacing the heated tweezers to prevent sticking materials if wax blocks are stuck on the tweezers; during embedding, the materials are required to be arranged in the required section rapidly, the materials are arranged singly at a distance of 1-1.2cm, and the embedded camellia oleifera flower bud wax strips are obtained after cooling for 24 hours at room temperature;
(7) wax trimming: cutting a shallow trench with the depth of about 1mm around the middle flower bud of the camellia oleifera flower bud embedded wax strip obtained in the step (6) by using a blade, slightly folding the wax strip by hand to split the wax strip into wax blocks, enabling each wax block to contain a flower bud in the center of the wax block, cutting and trimming the wax blocks into cubes by using the blade, and not exposing the flower buds outside, wherein at least 2-3mm of wax is left around the flower buds; dipping one end of a wood block with the side length of 2cm into molten paraffin, sticking the trimmed paraffin to the wood block, and ironing the joint of the paraffin and the wood block by using a heated paraffin melting shovel (or a flat-bottom spoon) to firmly stick the wood block and the wax; after the wax liquid is condensed, trimming redundant wax around the flower buds by using a blade, continuously cutting the flower buds with the depth of about 1mm each time until the flower buds are close to the flower buds from outside until the flower buds are 2mm away, horizontally cutting the flower buds into a cutter position closest to the flower buds from the deepest part of the lower cutter from outside to inside to ensure that the flower buds protrude into a cuboid shape, and finally trimming the part below the protruding part of the flower buds into a wedge shape by beveling the flower buds from a 45-degree angle on the side surface; obtaining trimmed camellia oleifera flower bud wax blocks;
(8) slicing: fixing the trimmed camellia oleifera flower bud wax block obtained in the step (7) on a sample fixer of a slicer, and adjusting the position of a wood block to enable the cut surface of the wax block to be parallel to a blade; installing a slicing knife on a slicing machine (model: KD-2258), adjusting the angle of the blade to 5 degrees, adjusting the thickness of the slice to 12 μm, opening a rotating wheel safety button to start slicing, picking up a wax belt cut into connected belts by a dissecting needle after 5 complete, clear and continuous slices are cut, placing the wax belt on a black paperboard, cutting off the wax belt by a scalpel, and writing material numbers on the paperboard to obtain the camellia flower bud slice wax belt;
(9) sheet sticking: loading a brand new plane glass slide into a 30-groove stainless steel glass slide rack, brushing the glass slide once with liquid detergent, washing the glass slide for 3 times with distilled water, soaking the glass slide for 3 seconds with 95% alcohol, and drying the glass slide for later use; weighing 25ml of egg white after removing yolk of 1 fresh egg, putting the egg white into a 100ml beaker, adding glycerol with the same volume, stirring and mixing completely for later use; dipping a small amount of sticky tablets with a cotton swab, uniformly coating the sticky tablets on the area of the frosted part of the cleaned glass slide, and baking the glass slide in a baking oven at the temperature of 45 ℃ for 2 hours; taking out the glass slide with the drying sticking piece, writing flower bud numbers on the frosted part, dripping distilled water, wherein the dripping area of the distilled water is not more than the area (about mu l) of the glass slide, taking the standard that the glass slide is fully dripped but does not flow out of the glass slide as a standard, then placing the camellia oleifera flower bud wax band in the center of the glass slide, sucking part of the distilled water by using a dropper, taking the volume of the distilled water which is enough for spreading the wax band but not easy to displace as a standard, and obtaining the camellia oleifera flower bud section wax band loaded on the glass slide;
(10) displaying and baking: flatly paving the glass slide with the distilled water and the camellia oleifera flower bud wax strips in the step (9), baking the glass slide in a 45 ℃ baking oven for 30min to enable the camellia oleifera flower bud sliced wax strips to be flat, and then continuously baking the glass slide in the 45 ℃ baking oven for 12h to enable the slices to be completely dried and tightly attached to the glass slide, so as to obtain camellia oleifera flower bud slices loaded on the glass slide;
(11) dewaxing: preparing 5 staining jars (length multiplied by width multiplied by height is 10.4cm multiplied by 9.4cm multiplied by 10cm), putting the camellia oleifera flower bud slices loaded on the glass slide obtained in the step (10) into a glass slide frame, immersing the slices in the staining jar filled with about 500ml of xylene solution for dewaxing for 20min, and then changing the xylene solution for dewaxing for 20min to obtain dewaxed camellia oleifera flower bud slices;
(12) rehydration: analytically pure xylene and absolute ethanol in a volume ratio of 1: 1, preparing 500ml of rehydration agent, and soaking the dewaxed camellia oleifera flower bud slices obtained in the step (11) in a staining jar filled with about 500ml of rehydration agent for rehydration for 10min to obtain pre-rehydrated camellia oleifera flower bud slices; sequentially adopting absolute ethyl alcohol to slice the pre-rehydrated camellia oleifera flower buds, replacing the absolute ethyl alcohol, replacing ethanol aqueous solutions with volume concentrations of 95%, 85% and 70% to soak for rehydration, wherein the soaking time of each stage is 5min, so as to obtain rehydrated camellia oleifera flower bud slices;
(13) dyeing: preparing 1% safranine staining agent by taking 5g safranine powder and 500ml of 50% ethanol aqueous solution in volume concentration, preparing 1% green staining agent by taking 5g green fixing powder and 500ml of 95% ethanol aqueous solution in volume concentration, immersing the reconstituted camellia oleifera flower bud slice in the step (12) in a staining jar filled with 500ml of 1% safranine staining agent for 4 hours, sequentially immersing in 85% and 95% ethanol aqueous solutions in volume concentration for 5min at each stage, and then replacing 1% green fixing staining agent for immersing for 13s to obtain a stained camellia oleifera flower bud slice;
(14) and (3) second dehydration: sequentially immersing the stained camellia oleifera flower bud slices obtained in the step (13) into an ethanol solution with the concentration volume of 95% and absolute ethanol, replacing the absolute ethanol, and immersing for 5min at each stage to obtain dehydrated stained camellia oleifera flower bud slices;
(15) and (3) second transparency: soaking the dehydrated and dyed camellia oleifera flower bud slices obtained in the step (14) in a mixed solution of xylene and absolute ethyl alcohol for 5min, then replacing with an analytically pure xylene solution for soaking for 5min, and replacing with the analytically pure xylene solution for soaking for 5min again to obtain transparent and dyed camellia oleifera flower bud slices; the mixed solution of the xylene and the absolute ethyl alcohol is analytically pure xylene and absolute ethyl alcohol according to the volume ratio of 1: 1 mixing the obtained mixed solution;
(16) when the slices are sealed, taking the transparent dyed camellia oleifera flower bud slices obtained in the step (15) out of xylene, placing the slices on white paper capable of absorbing water, and quickly dripping neutral resin on the edges of the slices before the xylene is dried; and lightly clamping the right side of the cover glass by using a small forceps, then contacting the left side of the cover glass with resin, then using a left hand to hold a dissecting needle to abut against the left side of the cover glass, controlling the forceps to slowly put down the cover glass by using the right hand to enable gum to spread along the putting-down direction of the cover glass and completely remove air, and after the cover glass is completely put down, slowly adjusting the position of the cover glass to enable the cover glass to completely cover tissues.
The paraffin section prepared in example 1 is observed under a microscope, and the specific result is shown in fig. 1, wherein fig. 1a is a camellia oleifera flower bud section under a 0.63-fold microscope, fig. 1b is a partial enlarged view (10-fold) of the camellia oleifera flower bud section of example 1, and fig. 1c is an enlarged view (40-fold) of the anther tissue of the camellia oleifera flower bud section of example 1, it can be seen that, in example 1, the tissues such as stamens and pistils in the camellia oleifera flower bud can be fully immersed into the stationary liquid by appropriately cutting the flower bud to expose the internal structure of the flower bud, the cellular morphological structure is complete, and in the steps of softening, dehydrating and waxing, each tissue of the camellia oleifera flower bud can achieve the expected effect after treatment, namely, the tissue hardness degree of each part of the camellia oleifera flower bud is equivalent, and the phenomenon that the sample is not completely fragmented and sliced into powder and silk due to dehydration and waxing is avoided. Therefore, the paraffin section of the camellia oleifera flower bud can clearly observe the structure of each tissue of the camellia oleifera flower bud, particularly the structure of anther, and the development period and the development condition of the flower bud can be quickly judged through the section.
Example 2
Compared with the embodiment 1, the difference lies in that the length of the collected camellia oleifera flower bud is 1.53cm, the paraffin section effect of the camellia oleifera flower bud in the embodiment is shown in fig. 2, wherein fig. 2a is a camellia oleifera flower bud section under a microscope of 0.63 times, fig. 2b is a partial enlarged view (10 times) of the camellia oleifera flower bud section in the embodiment 2, and fig. 2c is an enlarged view (40 times) of the anther tissue of the camellia oleifera flower bud section in the embodiment 2, and as can be seen from fig. 2, the section effect of the embodiment 2 is similar to that of the embodiment 1, and is not repeated, which indicates that the method provided by the invention is also applicable to larger flower buds and has a wide applicable range.
Comparative example 1
Step (1) of this comparative example is: collecting camellia oleifera flower buds with the length of 1.04cm by using scissors or a blade together with a flower stalk, directly and quickly putting the camellia oleifera flower buds into a fixing solution for fixing without any cutting treatment; the rest of the procedure was exactly the same as in example 1.
Observing the paraffin section prepared in the comparative example 1 under a microscope, wherein the specific result is shown in fig. 3, wherein fig. 3a is a camellia oleifera flower bud section under a microscope of 0.63 times, fig. 3b is a partial enlarged view (10 times) of the camellia oleifera flower bud section of the comparative example 1, and fig. 3c is an enlarged view (40 times) of anther tissue of the camellia oleifera flower bud section of the comparative example 1, and the paraffin section tissue of the camellia oleifera flower bud prepared in the comparative example 1 has obvious cracks, sepal tissue is deleted, displacement is overlapped, and petals are broken and deleted; the anther structure is not visible.
The analysis reason may be that the fixing liquid can not completely permeate into the inner tissue due to mutual wrapping and overlapping of the camellia oleifera flower bud tissue structures, the dehydration is incomplete, and the wax can not permeate into the inner tissue during wax dipping, so that the inner tissue is hard and brittle, and the slices can have the phenomenon of powder or silk and can not be sliced. In addition, due to the fact that the hardness of the external tissue is different from that of the internal tissue, cavities appear inside the slices, the outer-layer tissue sepals are cracked and displaced during slicing, the inner-layer tissue petals and the anther structure are incomplete, and the whole slice has cracks and the like.
Comparative example 2
The n-butanol in the first and second transparencies of this comparative example was replaced with xylene, and the n-butanol in the wax-dipped mixed solution was replaced with xylene, and the remaining steps were completely the same as in example 1.
The paraffin section prepared in the comparative example 2 is observed under a microscope, the clarity of the prepared paraffin section of the camellia oleifera flower bud is similar to that of the paraffin section prepared in the example 1, but xylene is volatilized and diffused in the air due to the large amount of xylene and the heating treatment, and irreversible damage is caused to the environment and experimenters.
In conclusion, the cutting treatment before the fixation of the camellia oleifera flower bud is beneficial to the operations of fixation, dehydration, wax dipping and the like of the flower bud tissue, so that the solution can be immersed in the flower bud tissue to fix and soften cells, the change of the morphological structures such as atrophy and the like caused by the decay of the cells can be prevented, the integrity of the cells is further maintained, and the rapid and complete fixation of the morphological structure of the camellia oleifera flower bud is beneficial; simultaneously the position is adjusted when still being favorable to the embedding, and the tangent plane is placed to the level down and is made the flower bud be on a parallel with the embedding box bottom surface during the embedding all the time, can cut the flower bud central authorities fast and contain the complete longitudinal section of structure such as stamen pistil during the section, and then conveniently observe the development change of the inside cell of flower bud and organizational structure. Next, n-butanol was used as a clearing agent in the clearing treatment. Because the toxicity of xylene in the traditional clearing agent is high, and the toxicity of n-butyl alcohol is far lower than that of xylene, the clearing agent has good clearing effect after being used as the clearing agent, smooth tissue, no obvious contraction and deformation phenomena, and the problem that the material becomes brittle due to overlong clearing operation time does not exist, so that the harm of xylene solution to experimenters is reduced to a great extent while the slicing result is ensured. And thirdly, the n-butyl alcohol paraffin melting mixed solution which is melted and mixed completely in advance is used as the wax dipping solution, the treatment is different from the traditional wax dipping method of gradually adding broken wax, the melting and mixing in advance are time-saving and labor-saving, and the wax dipping effect is good.
It is to be noted and understood that various modifications and improvements can be made to the invention described in detail above without departing from the spirit and scope of the invention as claimed in the appended claims. Accordingly, the scope of the claimed subject matter is not limited by any of the specific exemplary teachings provided.
The applicants hereby give notice that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. For those skilled in the art to which the invention pertains, several simple deductions or substitutions can be made without departing from the spirit of the invention, and all shall be considered as belonging to the protection scope of the invention.
Claims (8)
1. The method for preparing the paraffin section of the camellia oleifera flower bud is characterized by at least comprising the following steps:
(1) material taking, dividing and fixing: picking up camellia oleifera flower buds, stripping sepals, cutting off part of tissue of the camellia oleifera flower buds, fixing and softening;
(2) first dehydration and first transparency treatment: dehydrating the softened camellia oleifera flower buds by adopting gradient alcohol in sequence; then carrying out a clearing treatment, wherein the clearing agent at least comprises n-butanol;
(3) wax dipping: immersing the transparent camellia oleifera flower buds into an analytically pure n-butanol-paraffin mixed solution and a pure paraffin solution in sequence;
(4) embedding, wax trimming, slicing, sticking, spreading and baking the waxed camellia oleifera flower buds in sequence;
(5) then carrying out dewaxing, rehydration and dyeing treatment;
(6) second dehydration and second transparency treatment: dehydrating the dyed camellia oleifera flower buds by adopting gradient alcohol in sequence; then carrying out transparent treatment, wherein the transparent agent is dimethylbenzene;
(7) sealing: and sealing the slices with neutral resin, and drying to obtain paraffin sections of the camellia oleifera flower buds.
2. The production method according to claim 1, wherein in the step (1), the tissue of the volume of the camellia oleifera flower bud 1/3-1/5 is cut longitudinally;
preferably, the length of the exposed stamen after incision is taken as a standard:
for camellia oleifera flower buds smaller than 1.1cm, tissues with the volume of 1/5 camellia oleifera flower buds are longitudinally cut;
for camellia oleifera flower buds larger than 1.1cm, tissues with the volume of 1/3 camellia oleifera flower buds are longitudinally cut;
cutting camellia oleifera flower buds with the length of less than 0.5 cm.
3. The manufacturing method according to claim 1 or 2, wherein the transparent processing in the step (2) is specifically:
the dehydrated camellia oleifera flower buds are soaked in the first transparent agent and the second transparent agent in sequence and are replaced for at least 3 times, and the volume ratio of the dehydrated camellia oleifera flower buds to the solution of the transparent agents is more than 1: 10;
wherein, the first clearing agent is analytically pure n-butyl alcohol and analytically pure ethanol according to the volume ratio of 1: 1, mixing the solution;
and the second transparent agent is analytically pure butanol.
4. The production method according to any one of claims 1 to 3, wherein the analytically pure butanol paraffin mixture in step (3) is an analytically pure butanol paraffin mixture which is completely melted and mixed in advance in a high-temperature environment;
preferably, the temperature of the high-temperature environment is 75-85 ℃;
preferably, the mass-to-volume ratio of the analytically pure butanol to the paraffin is 1: 1 g/mL;
preferably, the step (3) of soaking further comprises the following steps:
baking at 55-65 deg.C, and soaking in liquid paraffin to obtain wax-soaked camellia oleifera flower bud;
preferably, the liquid paraffin has a temperature of 55 to 65 ℃.
5. The production method according to any one of claims 1 to 4, wherein the transparency treatment in the step (6) is specifically:
soaking the dehydrated and dyed camellia oleifera flower buds by using a third clearing agent and a fourth clearing agent in sequence;
wherein, the third clearing agent is analytically pure dimethylbenzene and analytically pure ethanol according to the volume ratio of 1: 1, mixing the solution;
the clearing agent four is analytically pure xylene.
6. Manufacturing method according to any one of claims 1 to 5, comprising at least the following steps:
(1) material taking, dividing and fixing: picking up camellia oleifera flower buds, stripping sepals, cutting off part of tissue of the camellia oleifera flower buds, fixing and softening; the length of the exposed stamen after cutting is taken as a standard:
for camellia oleifera flower buds smaller than 1.1cm, tissues with the volume of 1/5 camellia oleifera flower buds are longitudinally cut;
for camellia oleifera flower buds larger than 1.1cm, tissues with the volume of 1/3 camellia oleifera flower buds are longitudinally cut;
cutting camellia oleifera flower buds with the length of less than 0.5 cm;
(2) first dehydration and first transparency treatment: dehydrating the softened camellia oleifera flower buds by adopting gradient alcohol in sequence; the dehydrated camellia oleifera flower buds are soaked in the first transparent agent and the second transparent agent in sequence and are replaced for at least 3 times, and the volume ratio of the dehydrated camellia oleifera flower buds to the solution of the transparent agents is more than 1: 10;
wherein, the first clearing agent is analytically pure n-butyl alcohol and analytically pure ethanol according to the volume ratio of 1: 1, mixing the solution;
the second transparent agent is analytically pure butanol;
(3) wax dipping: soaking transparent camellia oleifera flower buds in an analytically pure butanol paraffin mixed solution; the analytically pure butanol paraffin mixed liquor is completely melted and mixed in advance in an environment of 75-85 ℃; the mass-volume ratio of the pure butanol to the paraffin is 1: 1 g/mL; baking at 55-65 deg.C, and soaking in 55-65 deg.C liquid paraffin to obtain wax-soaked camellia flower bud;
(4) embedding, wax trimming, slicing, sticking, spreading and baking the waxed camellia oleifera flower buds in sequence;
(5) then carrying out dewaxing, rehydration and dyeing treatment;
(6) second dehydration and second transparency treatment: dehydrating the dyed camellia oleifera flower buds by adopting gradient alcohol in sequence; soaking the dehydrated and dyed camellia oleifera flower buds by using a third clearing agent and a fourth clearing agent in sequence;
wherein, the third clearing agent is analytically pure dimethylbenzene and analytically pure ethanol according to the volume ratio of 1: 1, mixing the solution;
the clearing agent IV is analytically pure dimethylbenzene;
(7) sealing: and sealing the slices with neutral resin, and drying to obtain paraffin sections of the camellia oleifera flower buds.
7. A paraffin section of camellia oleifera flower buds, prepared by the method of any one of claims 1 to 5.
8. The use of the paraffin sections for camellia oleifera flower bud according to claim 7 in tissue organ and cell observation, study of camellia oleifera flower bud tissue-related pathology and camellia oleifera variety breeding.
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