CN111272511A - Method for making cucumber fruit thorn tumor paraffin slice - Google Patents
Method for making cucumber fruit thorn tumor paraffin slice Download PDFInfo
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- CN111272511A CN111272511A CN202010195555.1A CN202010195555A CN111272511A CN 111272511 A CN111272511 A CN 111272511A CN 202010195555 A CN202010195555 A CN 202010195555A CN 111272511 A CN111272511 A CN 111272511A
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- 235000013399 edible fruits Nutrition 0.000 title claims abstract description 101
- 235000010799 Cucumis sativus var sativus Nutrition 0.000 title claims abstract description 83
- 206010028980 Neoplasm Diseases 0.000 title claims abstract description 37
- 239000012188 paraffin wax Substances 0.000 title claims abstract description 30
- 238000000034 method Methods 0.000 title claims abstract description 25
- 244000299906 Cucumis sativus var. sativus Species 0.000 title 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 206
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- 239000000463 material Substances 0.000 claims abstract description 45
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- 238000006297 dehydration reaction Methods 0.000 claims abstract description 12
- 238000007598 dipping method Methods 0.000 claims abstract description 5
- 238000011161 development Methods 0.000 claims abstract description 4
- 235000019441 ethanol Nutrition 0.000 claims description 64
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 claims description 57
- 239000011521 glass Substances 0.000 claims description 49
- 230000006698 induction Effects 0.000 claims description 27
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Chemical compound C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 claims description 24
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 15
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- 238000004321 preservation Methods 0.000 claims description 9
- 238000004140 cleaning Methods 0.000 claims description 7
- 238000003860 storage Methods 0.000 claims description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 6
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 6
- 102000004169 proteins and genes Human genes 0.000 claims description 6
- 108090000623 proteins and genes Proteins 0.000 claims description 6
- 238000000926 separation method Methods 0.000 claims description 6
- 239000002023 wood Substances 0.000 claims description 6
- 230000008569 process Effects 0.000 claims description 5
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 claims description 3
- 102000002322 Egg Proteins Human genes 0.000 claims description 3
- 108010000912 Egg Proteins Proteins 0.000 claims description 3
- ABBQHOQBGMUPJH-UHFFFAOYSA-M Sodium salicylate Chemical compound [Na+].OC1=CC=CC=C1C([O-])=O ABBQHOQBGMUPJH-UHFFFAOYSA-M 0.000 claims description 3
- 229960000583 acetic acid Drugs 0.000 claims description 3
- XGGLLRJQCZROSE-UHFFFAOYSA-K ammonium iron(iii) sulfate Chemical compound [NH4+].[Fe+3].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O XGGLLRJQCZROSE-UHFFFAOYSA-K 0.000 claims description 3
- 239000007864 aqueous solution Substances 0.000 claims description 3
- 238000001816 cooling Methods 0.000 claims description 3
- 239000006059 cover glass Substances 0.000 claims description 3
- 235000014103 egg white Nutrition 0.000 claims description 3
- 210000000969 egg white Anatomy 0.000 claims description 3
- 230000003203 everyday effect Effects 0.000 claims description 3
- 238000011010 flushing procedure Methods 0.000 claims description 3
- 239000012362 glacial acetic acid Substances 0.000 claims description 3
- 235000011187 glycerol Nutrition 0.000 claims description 3
- 210000004932 little finger Anatomy 0.000 claims description 3
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- HLUCICHZHWJHLL-UHFFFAOYSA-N hematein Chemical compound C12=CC=C(O)C(O)=C2OCC2(O)C1=C1C=C(O)C(=O)C=C1C2 HLUCICHZHWJHLL-UHFFFAOYSA-N 0.000 description 2
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- 235000009849 Cucumis sativus Nutrition 0.000 description 1
- 244000303847 Lagenaria vulgaris Species 0.000 description 1
- 235000009797 Lagenaria vulgaris Nutrition 0.000 description 1
- 208000002193 Pain Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
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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
- G01N1/286—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
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- 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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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
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- G01N1/36—Embedding or analogous mounting of samples
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- 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
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- 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
- G01N2001/302—Stain compositions
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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
- G01N2001/305—Fixative compositions
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Abstract
The invention discloses a method for making cucumber fruit thorn tumor paraffin slice, which comprises the following steps: (1) selecting cucumber fruit burr materials in a proper development period, immediately putting the cucumber fruit burr materials into a container bottle filled with FAA fixing liquid, slightly shaking the container bottle for at least one time, and fixing for 30min after the cucumber fruit burr materials are settled on the liquid level; (2) pouring all FAA stationary liquid in the container bottle, adding new FAA stationary liquid into the container bottle again, sealing the bottle mouth with a sealing film, and fixing for at least 48 h; (3) then pouring all FAA stationary liquid in the container bottle again, adding ethanol with the concentration of 50% into the container bottle, and inducing for 1 h; (4) and finally, sequentially carrying out finishing dyeing, dehydration, transparence, wax dipping and embedding, slicing, sticking and unfolding, dewaxing, dyeing and sealing. The section obtained by the invention has the advantages of complete tissue, clear structure, strong operability and practicability, and is an effective method for observing the fruit thorn tumor anatomical structure.
Description
Technical Field
The invention relates to a method for making paraffin sections, in particular to a method for making cucumber fruit thorn tumor paraffin sections.
Background
The cucumber is a vegetable crop commonly cultivated in the world, is also the first crop produced in Chinese protected areas, and plays an important role in guaranteeing annual supply of vegetables. The cucumber fruits belong to the bottle gourd fruits, and the fruits have thorns but not uniform fruit tumors. The fruit tumor is a tumor-shaped protrusion on the surface of the cucumber fruit and is a characteristic trait of the cucumber. The existence of fruit tumor and the number of fruit thorn, the size of fruit thorn, the color of fruit peel and fruit thorn and the like belong to appearance quality characters, and are closely related to the economic value of cucumber. Compared with the cucumber variety with fruit tumor, the variety without fruit tumor has the advantages of smooth appearance, low pesticide residue, convenient cleaning, easy transportation, packaging and storage, and the like, and the market price of the variety is 2 to 3 times of that of the common cucumber. The research on the fruit oncotic character can promote the breeding process of cucumber quality, and the research can understand the cytological mechanism of the oncotic character by obtaining the operation technology of the oncotic paraffin section, thereby laying a foundation for the research on the molecular mechanism of the oncotic character.
Paraffin section is the most common microscopic section making technology, and has the characteristics of convenience, easy operation, low cost, permanent storage and the like, and is widely used for morphological and anatomical observation of plant materials, but different plants and different organs have different tissue structures and cannot be uniformly applied, so that the process of obtaining the paraffin section suitable for different plants, organs and tissues is especially important.
At present, the technical research applied to preparing cucumber fruit bur paraffin slices is less, the slicing effect is not ideal, and the main problems are that the cucumber burs are small and fragile, the slices are incomplete and the tissue structure is unclear. In order to better reveal the mechanism of the tuberculous morphosis, a method for preparing a paraffin section suitable for the tuberculous cucumber fruits is needed.
Disclosure of Invention
The invention aims to solve the problems and provides a method for making cucumber fruit thorn knot paraffin slices.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a manufacturing method of cucumber fruit thorn tumor paraffin slice comprises the following steps:
(1) selecting cucumber fruit burr materials in a proper development period, immediately putting the cucumber fruit burr materials into a container bottle filled with FAA fixing liquid, slightly shaking the container bottle for at least one time, and fixing for 30min after the cucumber fruit burr materials are settled on the liquid level;
(2) pouring all FAA stationary liquid in the container bottle, adding new FAA stationary liquid into the container bottle again, sealing the bottle mouth with a sealing film, and fixing for at least 48 h;
(3) then pouring all FAA stationary liquid in the container bottle again, adding ethanol with the concentration of 50% into the container bottle, and inducing for 1 h;
(4) and finally, sequentially carrying out finishing dyeing, dehydration, transparence, wax dipping and embedding, slicing, sticking and unfolding, dewaxing, dyeing and sealing.
In a preferred embodiment of the present invention, the FAA fixative solution is prepared from 38% formaldehyde aqueous solution, glacial acetic acid and 100% ethanol by volume ratio of 2: 1: 10 are mixed and prepared.
In a preferred embodiment of the present invention, the storage conditions in the step (2) are: storing at normal temperature or storing in a 4-degree refrigerator.
In a preferred embodiment of the present invention, the step of finishing in the step (3) is: putting the whole cucumber fruit bur material into a container bottle containing an ehrlich hematoxylin diluent for dyeing for 3-5 days, and slightly shaking the container bottle for 1-2 times every day, wherein the volume of the ehrlich hematoxylin diluent is 10-15 times of that of the cucumber fruit bur material.
In a preferred embodiment of the present invention, the dehydration step in the step (3) is: taking out cucumber fruit thorn tumor material from a container bottle containing an Eschka hematoxylin diluent, placing the cucumber fruit thorn tumor material into the container bottle containing 50% ethanol solution for room temperature induction for 30min, repeating the process again, changing the ethanol concentration in the container bottle to 60%, then conducting room temperature induction for 30min, then changing the ethanol concentration in the container bottle to 70%, then conducting room temperature induction for 30min, then changing the ethanol concentration in the container bottle to 80%, then conducting room temperature induction for 30min, changing the ethanol concentration in the container bottle to 90%, then conducting room temperature induction for 30min, then changing the ethanol concentration in the container bottle to 95%, then conducting room temperature induction for 30min, then changing the ethanol concentration in the container bottle to 100%, then conducting room temperature induction for 30min, then pouring out absolute ethanol in the container bottle, then guiding the absolute ethanol into a new absolute ethanol container bottle, then conducting room temperature induction for 30min again, and repeated 1 time.
In a preferred embodiment of the present invention, the step (3) of transparency is: after dehydration, pouring out absolute ethyl alcohol from the container bottle, changing the solution in the container bottle into 1/3 dimethylbenzene +2/3 absolute ethyl alcohol, inducing at room temperature for 30min, then changing the solution in the container bottle into 1/2 dimethylbenzene +1/2 absolute ethyl alcohol, inducing at room temperature for 30min, then changing the solution in the container bottle into 2/3 dimethylbenzene +1/3 absolute ethyl alcohol, inducing at room temperature for 30min, then changing the solution in the container bottle into pure dimethylbenzene, inducing at room temperature for 30min, repeating for 2 times, finally pouring out half of the dimethylbenzene in the container bottle, then adding half of wax chips, and standing at room temperature overnight.
In a preferred embodiment of the present invention, the steps of waxing and embedding in the step (3) are as follows: after the container bottle is transparent, the container bottle is placed in a 42 ℃ thermostat for heat preservation for 60min, then wax scraps are gradually added into the container bottle until the interior of the container bottle is filled, the heat preservation is needed for 60min after each wax scrap is added, then the mixed liquid in the container bottle is poured out, molten paraffin is added into the container bottle, then the container bottle is placed at the room temperature of 60 ℃ for induction for more than 4h and repeated for 6 times, then the embedding box is placed on a heat preservation table, the cucumber fruit burr material soaked with the wax and the paraffin are poured into the embedding box together, then the cucumber fruit burr material is slightly pulled by a preheating dissecting needle, the fruit burr material with upward fruit burrs and the whole cucumber fruit burr material are perpendicular to the bottom of the embedding box, then the embedding box is placed in a water tank for rapid cooling, and then the cucumber fruit burr material is stored in a refrigerator at 4 ℃ for later use.
In a preferred embodiment of the present invention, the slicing, sticking and spreading in step (3) comprises the following steps: opening the embedding box, using a single-sided blade to modify a wax block in the embedding box into a trapezoid shape, so that fruit spines of the cucumber fruit spines are positioned at the upper bottom and the fruit spines are positioned at the lower bottom, then adhering the wax block to a small wood block, fixing the small wood block on a paraffin slicer for slicing, setting the thickness of the sliced section to be 8 mu m, keeping the cucumber fruit spines at the vertical position of the blade during slicing, controlling the slicing speed to be uniform, and simultaneously using a writing brush to support a wax belt for continuous slicing;
after slicing, the cut wax tape is laid on white paper, the proper length is cut, a drop of protein sticking tablet is dropped on a glass slide, the glass slide is evenly smeared by a little finger, a drop of distilled water is added and evenly paved, the wax tape is clamped by a pair of tweezers and carefully laid on the glass slide, the wax tape is straightened by a dissecting needle, then the glass slide is placed on a slide-unfolding table to be unfolded for 1 to 2 days at the temperature of 42 ℃, and the protein sticking tablet is prepared by mixing 25ml of egg white, 25ml of glycerin and 0.5g of sodium salicylate.
In a preferred embodiment of the present invention, the dewaxing and dyeing step in step (3) is: putting the glass slide into pure dimethylbenzene for 2 times for dewaxing, wherein the first time is 20min and the second time is 15min, after dewaxing is finished, sequentially treating the glass slide with 1/2 dimethylbenzene, 1/2 absolute ethyl alcohol and absolute ethyl alcohol, repeating twice, wherein the treatment time is 3min each time, and then sequentially treating the glass slide with 95% ethyl alcohol, 90% ethyl alcohol, 85% ethyl alcohol, 75% ethyl alcohol, 60% ethyl alcohol and 50% ethyl alcohol, wherein the treatment time is 3min each time;
then cleaning the glass slide with distilled water for 6min, then counterstaining the glass slide with an Escholtzian hematoxylin diluent for 10min, then flushing the glass slide with running water for 15-20min, then carrying out color separation on the glass slide by adopting 4% ferric ammonium alum, then cleaning the glass slide with clear static microscopic color separation with distilled water for 6min, and removing redundant staining solution;
after counterstaining, the glass slide is sequentially treated according to 50% ethanol, 60% ethanol, 70% ethanol, 85% ethanol, 90% ethanol and 95% ethanol, each treatment time is 30s, then the glass slide is subjected to absolute ethanol treatment for 1min, and the treatment is repeated twice, then the glass slide is subjected to xylene treatment for 3min, and then the glass slide is subjected to xylene treatment for 10-30 min.
In a preferred embodiment of the present invention, the step of sealing in step (3) is: and (3) dropping a drop of neutral gum quickly after the glass slide is taken out of the xylene, wherein the operation time is as short as possible, the xylene is prevented from being volatilized completely, and then the cover glass is covered from one side, so that bubbles are prevented from influencing the observation quality.
The invention has the beneficial effects that:
the section obtained by the invention has the advantages of complete tissue, clear structure, strong operability and practicability, and is an effective method for observing the fruit thorn tumor anatomical structure. The invention provides a set of complete and efficient operation steps for cucumber fruit thorn tumor slicing technology, and provides technical reference for anatomical research of other organs and structure-like characters of cucumbers.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a diagram of the effect of cucumber stinging tumor tissue section in the microscope;
FIG. 2 is a microscopic experimental effect picture of a cucumber fruitless tumor large-thorn tissue section;
fig. 3 is a microscopic experimental effect picture of a cucumber fruitless tumor small fruit thorn tissue section.
Detailed Description
In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the invention is further explained below by combining the specific drawings.
The invention provides a method for making cucumber fruit thorn tumour paraffin slice, which comprises the following steps:
(1) selecting cucumber fruit burr materials in a proper development period, immediately putting the cucumber fruit burr materials into a container bottle filled with FAA fixing liquid, slightly shaking the container bottle for at least one time, and fixing for 30min after the cucumber fruit burr materials are settled on the liquid level;
(2) pouring all FAA stationary liquid in the container bottle, adding new FAA stationary liquid into the container bottle again, sealing the bottle mouth with a sealing film, and fixing for at least 48 h;
(3) then pouring all FAA stationary liquid in the container bottle again, adding ethanol with the concentration of 50% into the container bottle, and inducing for 1 h;
(4) and finally, sequentially carrying out finishing dyeing, dehydration, transparence, wax dipping and embedding, slicing, sticking and unfolding, dewaxing, dyeing and sealing.
The FAA stationary liquid adopted by the invention is prepared from 38% formaldehyde aqueous solution, glacial acetic acid and 100% ethanol in a volume ratio of 2: 1: 10, the slicing effect can be improved.
The storage conditions in the step (2) may be: the storage can be carried out at normal temperature or in a 4-degree refrigerator, and the storage can be determined according to actual conditions.
The finishing and dyeing step in the step (3) may specifically be: putting the whole cucumber fruit bur material into a container bottle containing an ehrlich hematoxylin diluent for dyeing for 3-5 days, and slightly shaking the container bottle for 1-2 times every day, wherein the volume of the ehrlich hematoxylin diluent is 10-15 times of that of the cucumber fruit bur material.
The dehydration step in the step (3) is: taking out cucumber fruit thorn tumor material from a container bottle containing an Eschka hematoxylin diluent, placing the cucumber fruit thorn tumor material into the container bottle containing 50% ethanol solution for room temperature induction for 30min, repeating the process again, changing the ethanol concentration in the container bottle to 60%, then conducting room temperature induction for 30min, then changing the ethanol concentration in the container bottle to 70%, then conducting room temperature induction for 30min, then changing the ethanol concentration in the container bottle to 80%, then conducting room temperature induction for 30min, changing the ethanol concentration in the container bottle to 90%, then conducting room temperature induction for 30min, then changing the ethanol concentration in the container bottle to 95%, then conducting room temperature induction for 30min, then changing the ethanol concentration in the container bottle to 100%, then conducting room temperature induction for 30min, then pouring out absolute ethanol in the container bottle, then guiding the absolute ethanol into a new absolute ethanol container bottle, then conducting room temperature induction for 30min again, and repeated 1 time.
In addition, as the hematein dye can be recycled, the dehydration time is long, and if the dehydration cannot be completed once, the cucumber fruit thorn tumor material can be stagnated in 70% ethanol for 12 hours.
The transparent step in the step (3) is specifically: after dehydration, pouring out absolute ethyl alcohol from the container bottle, changing the solution in the container bottle into 1/3 dimethylbenzene +2/3 absolute ethyl alcohol, inducing at room temperature for 30min, then changing the solution in the container bottle into 1/2 dimethylbenzene +1/2 absolute ethyl alcohol, inducing at room temperature for 30min, then changing the solution in the container bottle into 2/3 dimethylbenzene +1/3 absolute ethyl alcohol, inducing at room temperature for 30min, then changing the solution in the container bottle into pure dimethylbenzene, inducing at room temperature for 30min, repeating for 2 times, finally pouring out half of the dimethylbenzene in the container bottle, then adding half of wax chips, and standing at room temperature overnight.
The steps of the wax dipping and embedding in the step (3) are specifically as follows: after the container bottle is transparent, the container bottle is placed in a 42 ℃ thermostat for heat preservation for 60min, then wax scraps are gradually added into the container bottle until the interior of the container bottle is filled, the heat preservation is needed for 60min after each wax scrap is added, then the mixed liquid in the container bottle is poured out, molten paraffin is added into the container bottle, then the container bottle is placed at the room temperature of 60 ℃ for induction for more than 4h and repeated for 6 times, then the embedding box is placed on a heat preservation table, the cucumber fruit burr material soaked with the wax and the paraffin are poured into the embedding box together, then the cucumber fruit burr material is slightly pulled by a preheating dissecting needle, the fruit burr material with upward fruit burrs and the whole cucumber fruit burr material are perpendicular to the bottom of the embedding box, then the embedding box is placed in a water tank for rapid cooling, and then the cucumber fruit burr material is stored in a refrigerator at 4 ℃ for later use.
The slicing, sticking and spreading in the step (3) comprises the following steps: opening the embedding box, using a single-sided blade to modify a wax block in the embedding box into a trapezoid shape, so that fruit spines of the cucumber fruit spines are positioned at the upper bottom and the fruit spines are positioned at the lower bottom, then adhering the wax block to a small wood block, fixing the small wood block on a paraffin slicer for slicing, setting the thickness of the sliced section to be 8 mu m, keeping the cucumber fruit spines at the vertical position of the blade during slicing, controlling the slicing speed to be uniform, and simultaneously using a writing brush to support a wax belt for continuous slicing;
after slicing, the cut wax tape is laid on white paper, the proper length is cut, a drop of protein sticking tablet is dropped on a glass slide, the glass slide is evenly smeared by a little finger, a drop of distilled water is added and evenly paved, the wax tape is clamped by a pair of tweezers and carefully laid on the glass slide, the wax tape is straightened by a dissecting needle, then the glass slide is placed on a slide-unfolding table to be unfolded for 1 to 2 days at the temperature of 42 ℃, and the protein sticking tablet is prepared by mixing 25ml of egg white, 25ml of glycerin and 0.5g of sodium salicylate.
The dewaxing and dyeing in the step (3) comprises the following steps: the slides were dewaxed 2 times in pure xylene for a first time of 20min and a second time of 15 min.
Because the dimethylbenzene is volatile, the dimethylbenzene needs to be replaced for more than 5 times in order to ensure the dewaxing effect.
After the dewaxing by the pure xylene is carried out for two times, the non-target section can be removed through the initial examination by a microscope, and only the target section is subjected to subsequent dyeing, so that the processing time for obtaining the target section can be greatly shortened.
After dewaxing is finished, processing the glass slide with 1/2 xylene, 1/2 absolute ethyl alcohol and absolute ethyl alcohol in sequence, repeating the processing twice, wherein the processing time is 3min each time, and then processing with 95% ethyl alcohol, 90% ethyl alcohol, 85% ethyl alcohol, 75% ethyl alcohol, 60% ethyl alcohol and 50% ethyl alcohol in sequence, wherein the processing time is 3min each time;
then cleaning the glass slide with distilled water for 6min, then counterstaining the glass slide with an Escholtzian hematoxylin diluent for 10min, then flushing the glass slide with running water for 15-20min, then carrying out color separation on the glass slide by adopting 4% ferric ammonium alum, then cleaning the glass slide with clear static microscopic color separation with distilled water for 6min, and removing redundant staining solution;
after counterstaining, the glass slide is sequentially treated according to 50% ethanol, 60% ethanol, 70% ethanol, 85% ethanol, 90% ethanol and 95% ethanol, each treatment time is 30s, then the glass slide is subjected to absolute ethanol treatment for 1min, and the treatment is repeated twice, then the glass slide is subjected to xylene treatment for 3min, and then the glass slide is subjected to xylene treatment for 10-30 min.
The step of sealing the sheet in the step (3) is as follows: and (3) dropping a drop of neutral gum quickly after the glass slide is taken out of the xylene, wherein the operation time is as short as possible, the xylene is prevented from being volatilized completely, and then the cover glass is covered from one side, so that bubbles are prevented from influencing the observation quality.
Based on the method, the invention adopts the following three cucumber fruit thorn tumor materials for testing, and obtains the corresponding effects:
the first cucumber fruit thorn tumor material is a cucumber thorn tumor variety 7-10 days after blooming, and is cut along the periphery of the thorn tumor shape on the surface of the cucumber fruit in a minimized way, and the final effect is shown in figure 1;
the second cucumber fruit thorn tumor material is a large thorn variety without fruit tumor 7-10 days after blooming, and is cut along the periphery of the large thorn on the surface of the fruit in a minimized way, and the final effect is shown in figure 2;
the third cucumber fruit thorn tumor material is a small cucumber thorn variety without fruit tumor 7-10 days after blooming, and is cut along the periphery of the small thorn minimally on the surface of the cucumber fruit, so that a small amount of peel and pulp are contained, and the final effect is shown in figure 3.
As can be seen from FIGS. 1-3, the slices made by the method of the present invention are very complete and have very clear tissue structures.
The foregoing shows and describes the general principles and broad features of the present invention and advantages thereof. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (10)
1. A manufacturing method of cucumber fruit thorn tumor paraffin slice is characterized by comprising the following steps:
(1) selecting cucumber fruit burr materials in a proper development period, immediately putting the cucumber fruit burr materials into a container bottle filled with FAA fixing liquid, slightly shaking the container bottle for at least one time, and fixing for 30min after the cucumber fruit burr materials are settled on the liquid level;
(2) pouring all FAA stationary liquid in the container bottle, adding new FAA stationary liquid into the container bottle again, sealing the bottle mouth with a sealing film, and fixing for at least 48 h;
(3) then pouring all FAA stationary liquid in the container bottle again, adding ethanol with the concentration of 50% into the container bottle, and inducing for 1 h;
(4) and finally, sequentially carrying out finishing dyeing, dehydration, transparence, wax dipping and embedding, slicing, sticking and unfolding, dewaxing, dyeing and sealing.
2. The method for preparing cucumber fruit thorn tumor paraffin slice as claimed in claim 1, wherein the FAA fixing solution is prepared from 38% formaldehyde aqueous solution, glacial acetic acid and 100% ethanol by volume ratio of 2: 1: 10 are mixed and prepared.
3. The method for making cucumber fruit spines and paraffin slices as claimed in claim 1, wherein the storage conditions in the step (2) are as follows: storing at normal temperature or storing in a 4-degree refrigerator.
4. The method for making cucumber fruit spines and paraffin slices according to claim 1, wherein the step of finishing and dyeing in the step (3) comprises the following steps: putting the whole cucumber fruit bur material into a container bottle containing an ehrlich hematoxylin diluent for dyeing for 3-5 days, and slightly shaking the container bottle for 1-2 times every day, wherein the volume of the ehrlich hematoxylin diluent is 10-15 times of that of the cucumber fruit bur material.
5. The method for making cucumber fruit thorn tumour paraffin slice as claimed in claim 4, characterized in that the dehydration step in the step (3) is: taking out cucumber fruit thorn tumor material from a container bottle containing an Eschka hematoxylin diluent, placing the cucumber fruit thorn tumor material into the container bottle containing 50% ethanol solution for room temperature induction for 30min, repeating the process again, changing the ethanol concentration in the container bottle to 60%, then conducting room temperature induction for 30min, then changing the ethanol concentration in the container bottle to 70%, then conducting room temperature induction for 30min, then changing the ethanol concentration in the container bottle to 80%, then conducting room temperature induction for 30min, changing the ethanol concentration in the container bottle to 90%, then conducting room temperature induction for 30min, then changing the ethanol concentration in the container bottle to 95%, then conducting room temperature induction for 30min, then changing the ethanol concentration in the container bottle to 100%, then conducting room temperature induction for 30min, then pouring out absolute ethanol in the container bottle, then guiding the absolute ethanol into a new absolute ethanol container bottle, then conducting room temperature induction for 30min again, and repeated 1 time.
6. The manufacturing method of cucumber fruit thorn tumour paraffin section as claimed in claim 5, characterized in that, the transparent step in the step (3) is: after dehydration, pouring out absolute ethyl alcohol from the container bottle, changing the solution in the container bottle into 1/3 dimethylbenzene +2/3 absolute ethyl alcohol, inducing at room temperature for 30min, then changing the solution in the container bottle into 1/2 dimethylbenzene +1/2 absolute ethyl alcohol, inducing at room temperature for 30min, then changing the solution in the container bottle into 2/3 dimethylbenzene +1/3 absolute ethyl alcohol, inducing at room temperature for 30min, then changing the solution in the container bottle into pure dimethylbenzene, inducing at room temperature for 30min, repeating for 2 times, finally pouring out half of the dimethylbenzene in the container bottle, then adding half of wax chips, and standing at room temperature overnight.
7. The method for making cucumber fruit burr stone wax slices as claimed in claim 6, wherein the steps of waxing and embedding in the step (3) are as follows: after the container bottle is transparent, the container bottle is placed in a 42 ℃ thermostat for heat preservation for 60min, then wax scraps are gradually added into the container bottle until the interior of the container bottle is filled, the heat preservation is needed for 60min after each wax scrap is added, then the mixed liquid in the container bottle is poured out, molten paraffin is added into the container bottle, then the container bottle is placed at the room temperature of 60 ℃ for induction for more than 4h and repeated for 6 times, then the embedding box is placed on a heat preservation table, the cucumber fruit burr material soaked with the wax and the paraffin are poured into the embedding box together, then the cucumber fruit burr material is slightly pulled by a preheating dissecting needle, the fruit burr material with upward fruit burrs and the whole cucumber fruit burr material are perpendicular to the bottom of the embedding box, then the embedding box is placed in a water tank for rapid cooling, and then the cucumber fruit burr material is stored in a refrigerator at 4 ℃ for later use.
8. The method for making cucumber fruit spines and paraffin slices according to claim 7, wherein the slicing, sticking and spreading in the step (3) comprises the following steps: opening the embedding box, using a single-sided blade to modify a wax block in the embedding box into a trapezoid shape, so that fruit spines of the cucumber fruit spines are positioned at the upper bottom and the fruit spines are positioned at the lower bottom, then adhering the wax block to a small wood block, fixing the small wood block on a paraffin slicer for slicing, setting the thickness of the sliced section to be 8 mu m, keeping the cucumber fruit spines at the vertical position of the blade during slicing, controlling the slicing speed to be uniform, and simultaneously using a writing brush to support a wax belt for continuous slicing;
after slicing, the cut wax tape is laid on white paper, the proper length is cut, a drop of protein sticking tablet is dropped on a glass slide, the glass slide is evenly smeared by a little finger, a drop of distilled water is added and evenly paved, the wax tape is clamped by a pair of tweezers and carefully laid on the glass slide, the wax tape is straightened by a dissecting needle, then the glass slide is placed on a slide-unfolding table to be unfolded for 1 to 2 days at the temperature of 42 ℃, and the protein sticking tablet is prepared by mixing 25ml of egg white, 25ml of glycerin and 0.5g of sodium salicylate.
9. The method for making cucumber fruit spines and paraffin sections according to claim 8, wherein the dewaxing and dyeing in the step (3) comprises the following steps: putting the glass slide into pure dimethylbenzene for 2 times for dewaxing, wherein the first time is 20min and the second time is 15min, after dewaxing is finished, sequentially treating the glass slide with 1/2 dimethylbenzene, 1/2 absolute ethyl alcohol and absolute ethyl alcohol, repeating twice, wherein the treatment time is 3min each time, and then sequentially treating the glass slide with 95% ethyl alcohol, 90% ethyl alcohol, 85% ethyl alcohol, 75% ethyl alcohol, 60% ethyl alcohol and 50% ethyl alcohol, wherein the treatment time is 3min each time;
then cleaning the glass slide with distilled water for 6min, then counterstaining the glass slide with an Escholtzian hematoxylin diluent for 10min, then flushing the glass slide with running water for 15-20min, then carrying out color separation on the glass slide by adopting 4% ferric ammonium alum, then cleaning the glass slide with clear static microscopic color separation with distilled water for 6min, and removing redundant staining solution;
after counterstaining, the glass slide is sequentially treated according to 50% ethanol, 60% ethanol, 70% ethanol, 85% ethanol, 90% ethanol and 95% ethanol, each treatment time is 30s, then the glass slide is subjected to absolute ethanol treatment for 1min, and the treatment is repeated twice, then the glass slide is subjected to xylene treatment for 3min, and then the glass slide is subjected to xylene treatment for 10-30 min.
10. The method for making cucumber fruit burr stone wax slices as claimed in claim 9, wherein the sealing step in the step (3) is: and (3) dropping a drop of neutral gum quickly after the glass slide is taken out of the xylene, wherein the operation time is as short as possible, the xylene is prevented from being volatilized completely, and then the cover glass is covered from one side, so that bubbles are prevented from influencing the observation quality.
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Application publication date: 20200612 |