A method for preparing paraffin sections of mantis shrimp eyes

Abstract

The present invention provides a method for preparing paraffin sections of mantis shrimp eyeballs, comprising the following steps: step one: sampling; step two: fixation selection: fix with 4-6% paraformaldehyde at room temperature for 25-35 minutes; step three: dehydration treatment; step four: embedding the wax-impregnated tissue block with a modified paraffin wax; step five: continuously sectioning the sample using a microtome; step six: flattening the slices in a 37°C water bath; step seven: dewaxing and rehydrating after incubation at 37°C for 6-12 hours; step eight: after staining the sample, sealing the sample with a 50% sterile glycerol aqueous solution. The method for preparing paraffin sections of mantis shrimp eyeballs of the present invention comprises the improvement and optimization of procedures such as the dissection of the mantis shrimp eyeball shell, the selection of a fixative, dehydration, and waxing, breaking through the technical bottlenecks such as incomplete dehydration and waxing caused by the difficulty of dissecting the mantis shrimp eyeball due to the shell being difficult to dissect, damage to the blade due to the hard shell, and fragmentation of the slices.

Description

Technical Field

[0001] The present invention relates to the technical field of the preparation of sections, and particularly relates to a method for preparing paraffin sections of mantis shrimp eyes. Background Art

[0002] The paraffin section is the most widely used method in the conventional histological preparation techniques. Paraffin sections are not only used to observe the morphological structure of normal cell tissues, but also the main method for disciplines such as pathology and forensic medicine to study, observe, and judge the morphological changes of cell tissues, and have also been quite widely used in the research of many other disciplinary fields. The paraffin section method includes steps such as material selection, fixation, washing and dehydration, clearing, wax infiltration, embedding, sectioning and mounting, dewaxing, staining, dehydration, clearing, and coverslipping. Generally, it takes several days from material selection and fixation to coverslipping to make a glass slide specimen, but the specimen can be stored for a long time and used, and it is a permanent microscopic glass slide specimen.

[0003] The existing paraffin section preparation process is simple, using a simple dehydration process, and the dehydration is not thorough. At the same time, the paraffin embedding technology is simple, which leads to easy deformation, atrophy, and incomplete morphological preservation of the tissue after long-term placement, reducing the preparation efficiency of the sections. Based on this, the present invention further improves it; for example

[0004] Chinese Patent Document CN107976353A discloses a method for preparing paraffin sections of young roots of crops, including the following steps: material selection; microwave fixation; staining; microwave dehydration; microwave clearing; microwave wax infiltration; embedding; trimming the block; cementing; sectioning, spreading, and baking the sections; dewaxing; coverslipping and drying; wherein, the step of microwave fixation specifically includes the following operations: placing the plant tissue in a fixing solution, performing microwave treatment under the thawing file condition while water-bathing at 75°C, after 3 minutes, replacing the fixing solution at least twice, and performing microwave treatment under the thawing file condition while water-bathing at 75°C for 2 minutes each time after replacement;

[0005] However, this invention does not give a specific dehydration process, and the dehydration is not thorough. At the same time, the paraffin embedding technology is simple. After retrieval, it is found that the existing technology rarely studies the specific processes of dehydration and paraffin embedding, and the research technology is not mature. For the technology with high preparation efficiency, the present invention still needs further research and treatment. Summary of the Invention

[0006] Aiming at the defects of the existing technology, the purpose of the present invention is to provide a method for preparing paraffin sections of mantis shrimp eyes to solve the problems raised in the above background art.

[0007] The present invention adopts the following technical solutions to solve the technical problems:

[0008] The present invention provides a method for preparing paraffin sections of mantis shrimp eyes, including the following steps:

[0009] Step 1: Sample collection: Remove the hard ommatophore part, and retain the cornea part with low hardness but closest to the crystalline cone. Use clean ophthalmic scissors to cut off the eyeball along the base of the mantis shrimp ommatophore and soak it in filtered seawater. Use a corneal scissors and special ophthalmic forceps to anatomically remove the outer shell of the ommatophore part, and retain the outer shell of the cornea part;

[0010] Step 2: Selection of fixation: Fix at room temperature with 4 - 6% paraformaldehyde for 25 - 35 min;

[0011] Step 3: Dehydration treatment;

[0012] Step 4: Embed the tissue block after impregnation with modified paraffin;

[0013] Step 5: Use a microtome to continuously section the sample, with the section thickness of 4 μm;

[0014] Step 6: Flatten the sections in a 37°C water bath and spread them on polylysine - treated glass slides;

[0015] Step 7: After incubating at 37°C for 6 - 12 hours, dewax and rehydrate;

[0016] Step 8: After staining the sample, use a 50% glycerol sterile aqueous solution to cover - slip and preserve the sample.

[0017] Preferably, the specific operation steps of the dehydration treatment are as follows:

[0018] S01: Add 2 - 5% carbon nanotube modifier to the tissue sample in Step 3;

[0019] The preparation method of the carbon nanotube modifier is as follows:

[0020] S11: Feed carbon nanotubes into a hydrochloric acid solution with a concentration 5 - 10 times that of the carbon nanotubes, stir evenly, and then add 5 - 10% of the silane coupling agent KH560 and 1 - 4% of the sodium alginate solution based on the total amount of carbon nanotubes;

[0021] S12: Add graphene additive to the product of S11 according to a weight ratio of 1:6, stir thoroughly, and finally wash with water and dry to obtain the carbon nanotube modifier;

[0022] S02: Subsequently, dehydrate with 30% ethanol for 60 min, 50% ethanol for 60 min, 70% ethanol for 60 min, 95% ethanol for 60 min, and anhydrous ethanol twice for 30 min each; then dehydrate with xylene and anhydrous ethanol in a ratio of 1:1 for 5 min and xylene twice for 3 min each.

[0023] Preferably, the mass fractions of the hydrochloric acid solution and the sodium alginate solution are 2-5% and 10-15% respectively.

[0024] Preferably, the preparation method of the graphene additive is as follows: Add graphene into a sodium dodecyl sulfate solution with a mass fraction of 5-10% and a volume 2-5 times that of graphene, then add 1-5% lanthanum sulfate and 1-4% 4-aminophenylboronic acid, and continue to stir thoroughly to obtain the graphene additive.

[0025] Preferably, in the preparation of the graphene additive, the stirring speed is 1000-1500 r / min, the stirring time is 20-30 min, and the stirring temperature is 45-55 °C.

[0026] Preferably, the preparation method of the modified paraffin body is as follows:

[0027] S01: Feed bentonite into 3-5 times deionized water and stir to disperse it thoroughly, then wash and dry it with water.

[0028] S02: Subject the bentonite obtained in S01 to heat and cold treatment, and set it aside after the treatment.

[0029] S03: Add 5-10 parts of chitosan and 1-4 parts of sodium alginate into 10-15 parts of deionized water, then add 1-4 parts of sodium carboxymethylcellulose and 1-3 parts of yttrium nitrate, and stir evenly to obtain a modified solution.

[0030] S04: Stir the bentonite treated in S02 and the modified solution in S03 evenly according to a weight ratio of 1:5, wash and dry it with water, and finally mix it with paraffin according to a weight ratio of 1:7 to prepare the modified paraffin body.

[0031] Preferably, the specific steps of the heat and cold treatment of the bentonite are as follows:

[0032] First, heat the bentonite to 400-450 °C and keep it warm for 10-20 min, then cool it to 150-160 °C at a rate of 3-5 °C / min and keep it warm for 5-10 min, and finally cool it to room temperature with water.

[0033] Preferably, the temperature of the water cooling is 3-6 °C.

[0034] Preferably, the specific operation steps of the dewaxing and rehydration in Step 7 are as follows: Treat with xylene for 10 min, treat with a mixture of xylene and absolute ethanol (1:1) for 3 min, treat with 95% ethanol for 3 min, treat with 80% ethanol for 3 min, treat with 70% ethanol for 3 min, treat with 50% ethanol for 3 min, treat with 30% ethanol for 3 min, and treat with distilled water for 3 min.

[0035] Preferably, hematoxylin-eosin dye is used for staining the sample.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] The present invention provides a method for preparing paraffin sections of the eyes of Oratosquilla oratoria, including the improvement and optimization of procedures such as the dissection of the outer shell of the Oratosquilla oratoria eye, the selection of fixatives, dehydration, and infiltration with paraffin. It breaks through the technical bottlenecks such as incomplete dehydration and infiltration with paraffin caused by the difficult dissection of the outer shell of the Oratosquilla oratoria eye, the hard outer shell damaging the blade, and the generation of fragments during sectioning, as well as problems such as structural deformation, embrittlement of sections, and the presence of cavities that are prone to occur during the preparation of sections of small samples. Through this method, good section quality can be obtained, and the operation is simple and convenient. Description of the Drawings

[0038] Figure 1 It is the HE staining effect diagram of the paraffin section of the present invention. Detailed Embodiments

[0039] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts shall fall within the protection scope of the present invention.

[0040] Refer to Figure 1 , a method for preparing paraffin sections of the eyes of Oratosquilla oratoria, including the following steps:

[0041] Step 1: Material collection: Remove the harder eye stalk part, retain the cornea part with lower hardness but closest to the crystalline cone, cut off the Oratosquilla oratoria eye along the base of the eye stalk with clean ophthalmic scissors and soak it in filtered seawater. Use a corneal scissors and special ophthalmic forceps to dissect and remove the outer shell of the eye stalk part, and retain the outer shell of the cornea part;

[0042] Step 2: Selection of fixation: Fix at room temperature with 4 - 6% paraformaldehyde for 25 - 35 minutes;

[0043] Step 3: Dehydration treatment;

[0044] Step 4: Embed the infiltrated tissue block with a modified paraffin body;

[0045] Step 5: Continuously section the sample using a microtome, with the section thickness of 4 μm;

[0046] Step 6: Flatten the sections in a 37°C water bath and lay them flat on a polylysine-treated glass slide;

[0047] Step 7: After incubating at 37°C for 6 - 12 hours, perform dewaxing and rehydration;

[0048] Step 8: After staining the sample, use 50% glycerol sterile aqueous solution to seal the sample for preservation.

[0049] The specific operation steps of the dehydration process in this embodiment are:

[0050] S01: adding 2-5% carbon nanotube modifier to the tissue sample in step 3;

[0051] The preparation method of the carbon nanotube modifier is as follows:

[0052] S11: adding the carbon nanotubes to a 5-10 times hydrochloric acid solution, stirring evenly, and then adding 5-10% of the total amount of the carbon nanotubes, a silane coupling agent KH560, and 1-4% of a sodium alginate solution;

[0053] S12: adding the graphene additive to the product of S11 at a weight ratio of 1:6, stirring thoroughly, and finally washing with water and drying to obtain a carbon nanotube modifier;

[0054] S02: Dehydrate with 30% ethanol for 60 min, 50% ethanol for 60 min, 70% ethanol for 60 min, 95% ethanol for 60 min, and anhydrous ethanol twice for 30 min each; then dehydrate with xylene and anhydrous ethanol in a ratio of 1:1 for 5 min, and dehydrate with xylene twice for 3 min each time.

[0055] The mass fractions of the hydrochloric acid solution and the sodium alginate solution in this embodiment are 2-5% and 10-15% respectively.

[0056] The preparation method of the graphene additive of this embodiment is as follows: adding graphene to 2-5 times the mass fraction of 5-10% sodium dodecyl sulfate solution, then adding 1-5% lanthanum sulfate and 1-4% 4-aminophenylboric acid, and continuing to stir thoroughly to obtain the graphene additive.

[0057] In the preparation of the graphene additive of this embodiment, the stirring speed is 1000-1500 r / min, the stirring time is 20-30 min, and the stirring temperature is 45-55°C.

[0058] The preparation method of the modified paraffin wax of this embodiment is as follows:

[0059] S01: Add bentonite to 3-5 times the amount of deionized water and stir to disperse thoroughly, then wash and dry;

[0060] S02: heat and cool the bentonite in S01 until the treatment is complete and the bentonite is ready for use;

[0061] S03: Add 5 - 10 parts of chitosan and 1 - 4 parts of sodium alginate into 10 - 15 parts of deionized water, then add 1 - 4 parts of sodium carboxymethyl cellulose and 1 - 3 parts of yttrium nitrate, and stir evenly to obtain a modified solution;

[0062] S04: Stir evenly the bentonite treated in S02 and the modified solution of S03 according to a weight ratio of 1:5, wash with water and dry, and finally mix and prepare with paraffin according to a weight ratio of 1:7 to obtain a modified paraffin body.

[0063] The specific steps for the heat - cold treatment of bentonite in this example are as follows:

[0064] First, heat the bentonite to 400 - 450 °C, keep it warm for 10 - 20 min, then cool it to 150 - 160 °C at a rate of 3 - 5 °C / min, keep it warm for 5 - 10 min, and finally cool it to room temperature with water.

[0065] The temperature of water cooling in this example is 3 - 6 °C.

[0066] The specific operation steps for dewaxing and rehydration in step seven of this example are: treat with xylene for 10 min, treat with xylene: absolute ethanol = 1:1 for 3 min, treat with 95% ethanol for 3 min, treat with 80% ethanol for 3 min; treat with 70% ethanol for 3 min, treat with 50% ethanol for 3 min, treat with 30% ethanol for 3 min, and treat with distilled water for 3 min.

[0067] In this example, hematoxylin - eosin dye is used to stain the sample.

[0068] Example 1.

[0069] A method for preparing paraffin sections of mantis shrimp eyes, comprising the following steps:

[0070] Step 1: Specimen collection: Remove the harder eyestalk part, retain the cornea part with lower hardness but closest to the crystalline cone. Cut off the mantis shrimp eye with clean ophthalmic scissors along the base of the mantis shrimp eyestalk and soak it in filtered seawater. Use a corneal scissors and special ophthalmic forceps to anatomically remove the outer shell of the eyestalk part, and retain the outer shell of the cornea part;

[0071] Step 2: Fixative selection: Fix at room temperature with 4% paraformaldehyde for 25 min;

[0072] Step 3: Dehydration treatment;

[0073] Step 4: Embed the tissue block after impregnation with wax using the modified paraffin body;

[0074] Step 5: Continuously section the sample using a microtome, with the section thickness being 4 μm;

[0075] Step 6: Flatten the slices in a 37°C water bath and place them on poly-lysine-treated slides;

[0076] Step 7: Incubate at 37°C for 6 hours before dewaxing and rehydrating;

[0077] Step 8: After staining the sample, use 50% glycerol sterile aqueous solution to seal the sample for preservation.

[0078] The specific operation steps of the dehydration process in this embodiment are:

[0079] S01: adding 2% carbon nanotube modifier to the tissue sample in step 3;

[0080] The preparation method of the carbon nanotube modifier is as follows:

[0081] S11: adding the carbon nanotubes to a 5-fold hydrochloric acid solution, stirring evenly, and then adding 5% of the total amount of the carbon nanotubes, a silane coupling agent KH560, and 1% of a sodium alginate solution;

[0082] S12: adding the graphene additive to the product of S11 at a weight ratio of 1:6, stirring thoroughly, and finally washing with water and drying to obtain a carbon nanotube modifier;

[0083] S02: Dehydrate with 30% ethanol for 60 min, 50% ethanol for 60 min, 70% ethanol for 60 min, 95% ethanol for 60 min, and anhydrous ethanol twice for 30 min each; then dehydrate with xylene and anhydrous ethanol in a ratio of 1:1 for 5 min, and dehydrate with xylene twice for 3 min each time.

[0084] The mass fractions of the hydrochloric acid solution and the sodium alginate solution in this embodiment are 2% and 10% respectively.

[0085] The preparation method of the graphene additive of this embodiment is as follows: adding graphene to 2 times the mass fraction of 5% sodium dodecyl sulfate solution, then adding 1% lanthanum sulfate and 1% 4-aminophenylboric acid, and continuing to stir thoroughly to obtain the graphene additive.

[0086] In the preparation of the graphene additive of this embodiment, the stirring speed is 1000 r / min, the stirring time is 20 min, and the stirring temperature is 45°C.

[0087] The preparation method of the modified paraffin wax of this embodiment is as follows:

[0088] S01: Add bentonite to 3 times the amount of deionized water and stir to disperse thoroughly, then wash and dry;

[0089] S02: heat and cool the bentonite in S01 until the treatment is complete and the bentonite is ready for use;

[0090] S03: Add 5 parts of chitosan and 1 part of sodium alginate into 10 parts of deionized water, then add 1 part of sodium carboxymethylcellulose and 1 part of yttrium nitrate, and stir evenly to obtain a modified solution.

[0091] S04: Stir evenly the bentonite treated by S02 and the modified solution of S03 according to a weight ratio of 1:5, wash with water and dry, and finally mix and prepare with paraffin according to a weight ratio of 1:7 to obtain a modified paraffin body.

[0092] The specific steps for the thermal and cold treatment of bentonite in this example are as follows:

[0093] First, heat the bentonite to 400 °C, keep it warm for 10 min, then cool it to 150 °C at a rate of 3 °C / min, keep it warm for 5 min, and finally cool it to room temperature with water.

[0094] The water cooling temperature in this example is 3 °C.

[0095] The specific operation steps for dewaxing and rehydration in step seven of this example are as follows: treat with xylene for 10 min, treat with xylene: absolute ethanol = 1:1 for 3 min, treat with 95% ethanol for 3 min, treat with 80% ethanol for 3 min, treat with 70% ethanol for 3 min, treat with 50% ethanol for 3 min, treat with 30% ethanol for 3 min, and treat with distilled water for 3 min.

[0096] In this example, hematoxylin-eosin dye is used to stain the sample.

[0097] Example 2.

[0098] A method for preparing paraffin sections of mantis shrimp eyes includes the following steps:

[0099] Step 1: Material collection: Remove the hard eyestalk part, retain the cornea part with low hardness but closest to the crystalline cone, cut off the mantis shrimp eye with clean ophthalmic scissors along the base of the mantis shrimp eyestalk and soak it in filtered seawater, and use a corneal scissors and ophthalmic special forceps to anatomically remove the outer shell of the eyestalk part, retaining the outer shell of the cornea part.

[0100] Step 2: Selection of fixation: Fix at room temperature with 6% paraformaldehyde for 35 min.

[0101] Step 3: Dehydration treatment;

[0102] Step 4: Embed the tissue block after impregnation with wax using the modified paraffin body.

[0103] Step 5: Continuously section the sample using a microtome, with the section thickness being 4 μm.

[0104] Step 6: Flatten the slices in a 37°C water bath and place them on poly-lysine-treated slides;

[0105] Step 7: Incubate at 37°C for 12 hours before dewaxing and rehydrating;

[0106] Step 8: After staining the sample, use 50% glycerol sterile aqueous solution to seal the sample for preservation.

[0107] The specific operation steps of the dehydration process in this embodiment are:

[0108] S01: adding 5% carbon nanotube modifier to the tissue sample in step 3;

[0109] The preparation method of the carbon nanotube modifier is as follows:

[0110] S11: adding the carbon nanotubes to a 10-fold hydrochloric acid solution, stirring evenly, and then adding 10% of the total amount of the carbon nanotubes, a silane coupling agent KH560, and 4% of a sodium alginate solution;

[0111] S12: adding the graphene additive to the product of S11 at a weight ratio of 1:6, stirring thoroughly, and finally washing with water and drying to obtain a carbon nanotube modifier;

[0112] S02: Dehydrate with 30% ethanol for 60 min, 50% ethanol for 60 min, 70% ethanol for 60 min, 95% ethanol for 60 min, and anhydrous ethanol twice for 30 min each; then dehydrate with xylene and anhydrous ethanol in a ratio of 1:1 for 5 min, and dehydrate with xylene twice for 3 min each time.

[0113] The mass fractions of the hydrochloric acid solution and the sodium alginate solution in this embodiment are 5% and 15% respectively.

[0114] The preparation method of the graphene additive of this embodiment is as follows: adding graphene to 5 times the mass fraction of 10% sodium dodecyl sulfate solution, then adding 5% lanthanum sulfate and 4% 4-aminophenylboric acid, and continuing to stir thoroughly to obtain the graphene additive.

[0115] In the preparation of the graphene additive of this embodiment, the stirring speed is 1500 r / min, the stirring time is 30 min, and the stirring temperature is 55°C.

[0116] The preparation method of the modified paraffin wax of this embodiment is as follows:

[0117] S01: Add bentonite to 3-5 times the amount of deionized water and stir to disperse thoroughly, then wash and dry;

[0118] S02: heat and cool the bentonite in S01 until the treatment is complete and the bentonite is ready for use;

[0119] S03: Add 10 parts of chitosan and 4 parts of sodium alginate into 15 parts of deionized water, then add 4 parts of sodium carboxymethylcellulose and 3 parts of yttrium nitrate, and stir evenly to obtain a modified solution.

[0120] S04: Stir the bentonite treated by S02 and the modified solution of S03 evenly according to the weight ratio of 1:5, wash with water and dry, and finally mix and prepare with paraffin according to the weight ratio of 1:7 to obtain a modified paraffin body.

[0121] The specific steps of the thermal and cold treatment of bentonite in this example are as follows:

[0122] First, heat the bentonite to 450 °C, keep it warm for 20 min, then cool it to 160 °C at a rate of 5 °C / min, keep it warm for 10 min, and finally cool it to room temperature with water.

[0123] The water cooling temperature in this example is 6 °C.

[0124] The specific operation steps of dewaxing and rehydration in step seven of this example are as follows: Treat with xylene for 10 min, treat with xylene: absolute ethanol = 1:1 for 3 min, treat with 95% ethanol for 3 min, treat with 80% ethanol for 3 min; treat with 70% ethanol for 3 min, treat with 50% ethanol for 3 min, treat with 30% ethanol for 3 min, and treat with distilled water for 3 min.

[0125] In this example, hematoxylin-eosin dye is used to stain the sample.

[0126] Example 3.

[0127] A method for preparing paraffin sections of mantis shrimp eyes includes the following steps:

[0128] Step 1: Material collection: Remove the hard ommatophore part, retain the cornea part with low hardness but closest to the crystalline cone, cut off the mantis shrimp eye with clean ophthalmic scissors along the base of the mantis shrimp ommatophore and soak it in filtered seawater, and use a corneal scissors and ophthalmic special forceps to anatomically remove the outer shell of the ommatophore part and retain the outer shell of the cornea part.

[0129] Step 2: Fixative selection: Fix at room temperature with 5% paraformaldehyde for 30 min.

[0130] Step 3: Dehydration treatment;

[0131] Step 4: Embed the tissue block after impregnation with wax using the modified paraffin body.

[0132] Step 5: Continuously section the sample using a microtome, and the section thickness is 4 μm.

[0133] Step 6: Flatten the slices in a 37°C water bath and place them on poly-lysine-treated slides;

[0134] Step 7: Incubate at 37°C for 10 hours before dewaxing and rehydrating;

[0135] Step 8: After staining the sample, use 50% glycerol sterile aqueous solution to seal the sample for preservation.

[0136] The specific operation steps of the dehydration process in this embodiment are:

[0137] S01: adding 3.5% carbon nanotube modifier to the tissue sample in step 3;

[0138] The preparation method of the carbon nanotube modifier is as follows:

[0139] S11: adding the carbon nanotubes to a 7.5-fold hydrochloric acid solution, stirring evenly, and then adding 7.5% of the total amount of carbon nanotubes, a silane coupling agent KH560, and 2.5% of a sodium alginate solution;

[0140] S12: adding the graphene additive to the product of S11 at a weight ratio of 1:6, stirring thoroughly, and finally washing with water and drying to obtain a carbon nanotube modifier;

[0141] S02: Dehydrate with 30% ethanol for 60 min, 50% ethanol for 60 min, 70% ethanol for 60 min, 95% ethanol for 60 min, and anhydrous ethanol twice for 30 min each; then dehydrate with xylene and anhydrous ethanol in a ratio of 1:1 for 5 min, and dehydrate with xylene twice for 3 min each time.

[0142] The mass fractions of the hydrochloric acid solution and the sodium alginate solution in this embodiment are 3.5% and 12.5% respectively.

[0143] The preparation method of the graphene additive of this embodiment is as follows: adding graphene to 3.5 times the mass fraction of 7.5% sodium dodecyl sulfate solution, then adding 3% lanthanum sulfate and 2% 4-aminophenylboric acid, and continuing to stir thoroughly to obtain the graphene additive.

[0144] In the preparation of the graphene additive of this embodiment, the stirring speed is 1250 r / min, the stirring time is 25 min, and the stirring temperature is 50°C.

[0145] The preparation method of the modified paraffin wax of this embodiment is as follows:

[0146] S01: Add bentonite to 4 times the amount of deionized water and stir to disperse thoroughly, then wash and dry;

[0147] S02: Heat and then cool the bentonite obtained in S01. After the treatment, set it aside for later use.

[0148] S03: Add 7.5 parts of chitosan and 2.5 parts of sodium alginate into 12.5 parts of deionized water. Then add 2.5 parts of sodium carboxymethylcellulose and 2 parts of yttrium nitrate, and stir evenly to obtain a modified solution.

[0149] S04: Stir evenly the bentonite treated in S02 and the modified solution in S03 according to a weight ratio of 1:5. Wash with water and dry. Finally, mix and prepare with paraffin according to a weight ratio of 1:7 to obtain a modified paraffin body.

[0150] The specific steps for the heat and cool treatment of the bentonite in this example are as follows:

[0151] First, heat the bentonite to 425 °C and keep it warm for 15 min. Then, cool it to 150 - 160 °C at a rate of 4 °C / min and keep it warm for 5 - 10 min. Finally, cool it with water to room temperature.

[0152] The water cooling temperature in this example is 4.5 °C.

[0153] The specific operation steps for dewaxing and rehydration in step seven of this example are as follows: Treat with xylene for 10 min, treat with a mixture of xylene and absolute ethanol (1:1) for 3 min, treat with 95% ethanol for 3 min, treat with 80% ethanol for 3 min, treat with 70% ethanol for 3 min, treat with 50% ethanol for 3 min, treat with 30% ethanol for 3 min, and treat with distilled water for 3 min.

[0154] In this example, hematoxylin - eosin dye is used to stain the sample.

[0155] Example 4.

[0156] A method for preparing paraffin sections of mantis shrimp eyes includes the following steps:

[0157] Step 1: Sampling: Remove the hard eyestalk part, retain the cornea part with low hardness but closest to the crystalline cone. Cut off the mantis shrimp eye with clean ophthalmic scissors along the base of the mantis shrimp eyestalk and soak it in filtered seawater. Use a corneal scissor and special ophthalmic forceps to anatomically remove the outer shell of the eyestalk part, and retain the outer shell of the cornea part.

[0158] Step 2: Fixative selection: Fix at room temperature with 5% paraformaldehyde for 27 min.

[0159] Step 3: Dehydration treatment;

[0160] Step 4: Embed the tissue block after impregnation with wax using the modified paraffin body;

[0161] Step 5: Use a microtome to continuously section the sample, with the section thickness being 4 μm;

[0162] Step 6: Flatten the sections in a 37°C water bath and lay them flat on polylysine-treated glass slides.

[0163] Step 7: After incubating at 37°C for 7 hours, dewax and rehydrate.

[0164] Step 8: After staining the samples, cover-slip and preserve the samples with a 50% sterile aqueous glycerol solution.

[0165] The specific operating steps of the dehydration treatment in this example are as follows:

[0166] S01: Add a 3% carbon nanotube modifier to the tissue sample in Step 3.

[0167] The preparation method of the carbon nanotube modifier is as follows:

[0168] S11: Feed the carbon nanotubes into 6 times the hydrochloric acid solution, stir evenly, and then add 6% of the silane coupling agent KH560 and 2% of the sodium alginate solution based on the total amount of carbon nanotubes.

[0169] S12: Add the graphene additive to the product of S11 according to a weight ratio of 1:6, stir thoroughly, and finally wash with water and dry to obtain the carbon nanotube modifier.

[0170] S02: Subsequently, dehydrate with 30% ethanol for 60 min, with 50% ethanol for 60 min, with 70% ethanol for 60 min, with 95% ethanol for 60 min, and with absolute ethanol twice for 30 min each; then dehydrate with xylene and absolute ethanol in a 1:1 ratio for 5 min and with xylene twice for 3 min each.

[0171] The mass fractions of the hydrochloric acid solution and the sodium alginate solution in this example are 3% and 12% respectively.

[0172] The preparation method of the graphene additive in this example is: Add graphene to 3 times the sodium dodecyl sulfate solution with a mass fraction of 6%, then add 2% of lanthanum sulfate and 2% of 4-aminophenylboronic acid, and continue to stir thoroughly to obtain the graphene additive.

[0173] In the preparation of the graphene additive in this example, the stirring speed for thorough stirring is 1200 r / min, the stirring time is 22 min, and the stirring temperature is 46°C.

[0174] The preparation method of the modified paraffin body in this example is as follows:

[0175] S01: Feed bentonite into 4 times deionized water, stir and disperse thoroughly, and finally wash with water and dry.

[0176] S02: Heat and then cool the bentonite in S01. After the treatment is completed, set it aside for later use.

[0177] S03: Add 6 parts of chitosan and 2 parts of sodium alginate to 12 parts of deionized water. Then add 2 parts of sodium carboxymethylcellulose and 2 parts of yttrium nitrate, and stir evenly to obtain a modified liquid.

[0178] S04: Stir evenly the bentonite treated in S02 and the modified liquid in S03 according to a weight ratio of 1:5, wash with water and dry. Finally, mix and prepare it with paraffin according to a weight ratio of 1:7 to obtain a modified paraffin body.

[0179] The specific steps for the heat and cool treatment of the bentonite in this example are as follows:

[0180] First, heat the bentonite to 420 °C, keep it warm for 12 min, then cool it to 155 °C at a rate of 4 °C / min, keep it warm for 6 min, and finally cool it to room temperature with water.

[0181] The water cooling temperature in this example is 4 °C.

[0182] The specific operation steps for dewaxing and rehydration in step seven of this example are as follows: Treat with xylene for 10 min, treat with xylene: absolute ethanol = 1:1 for 3 min, treat with 95% ethanol for 3 min, treat with 80% ethanol for 3 min, treat with 70% ethanol for 3 min, treat with 50% ethanol for 3 min, treat with 30% ethanol for 3 min, and treat with distilled water for 3 min.

[0183] In this example, hematoxylin-eosin dye is used to stain the sample.

[0184] The paraffin sections prepared by the methods of Examples 1-4 are not prone to deformation, shrinkage, and their morphology remains intact after long-term placement.

[0185] Comparative Example 1.

[0186] The difference from Example 3 is that no carbon nanotube modifier is added.

[0187] Comparative Example 2.

[0188] The difference from Example 3 is that the modified paraffin body is replaced with paraffin.

[0189] Comparative Example 3.

[0190] The difference from Example 3 is that no modified liquid treatment is carried out during the preparation of the modified paraffin body.

[0191] Comparative Example 4.

[0192] The difference from Example 3 is that no graphene additive is added during the preparation of the carbon nanotube modifier.

[0193] The performance measurement results of Examples 1-4 and Comparative Examples 1-4 are as follows

[0194] After the sample tissue was placed for 1 month, observe the tissue condition Example 1 The sample tissue was intact and not deformed, and there was no obvious change in the tissue condition Example 2 The sample tissue was intact and not deformed, and there was no obvious change in the tissue condition Example 3 The sample tissue was intact and not deformed, and there was no obvious change in the tissue condition Example 4 The sample tissue was intact and not deformed, and there was no obvious change in the tissue condition Comparative Example 1 The sample tissue was deformed, damaged, and the cell structure was unclear Comparative Example 2 The sample tissue was deformed, damaged, and the cell structure was unclear Comparative Example 3 The cell structure of the sample tissue was unclear and not deformed Comparative Example 4 The cell structure of the sample tissue was unclear and not deformed

[0195] It can be concluded from Examples 1-4 and Comparative Examples 1-4 that when using a carbon nanotube modifier to cooperate with a modified paraffin body and optimizing the process, the tissue morphology of the sample is excellent, without morphological changes, and the tissue cells are clear;

[0196] The innovation of the present invention lies in: treating the tissue sample with a carbon nanotube modifier during dehydration treatment, treating the carbon nanotubes with sodium alginate solution, hydrochloric acid and silane coupling agent to improve its surface activity and dispersion effect; after graphene is optimized, the sheet structure is active, cooperating with the dispersion of carbon nanotubes to promote higher dehydration efficiency of the tissue sample, and bentonite is dispersed with deionized water, and the interlayer spacing is expanded through heat and cold treatment, and the activity is higher after the treatment with the modified liquid, which is more suitable for cooperating with paraffin. Through paraffin cooperation, its sealing effect is improved. After the process is optimized and modified, the dehydration and embedding effects are enhanced; the prepared sections are easier to preserve, not easy to shrink, the sample tissue is preserved more completely, and after the sectioning process is optimized and improved, it has a significant improvement effect compared with the prior art.

[0197] For those skilled in the art, it is obvious that the present invention is not limited to the details of the above exemplary embodiments, and without departing from the spirit or basic characteristics of the present invention, the present invention can be implemented in other specific forms. Therefore, from any point of view, the embodiments should be regarded as exemplary and non-limiting. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be included in the present invention.

[0198] In addition, it should be understood that although this specification is described according to embodiments, not every embodiment only contains an independent technical solution. This narrative way of the specification is only for clarity. Those skilled in the art should regard the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for preparing paraffin sections of mantis shrimp eyes, characterized in that, It includes the following steps: Step 1: Material selection: Remove the harder eyestalk part, retain the cornea part with low hardness but closest to the photoreceptor cells. Cut off the mantis shrimp eyeball at the base of the eyestalk with clean ophthalmic scissors and soak it in filtered seawater. Use a corneal scissors and special ophthalmic forceps to anatomically remove the outer shell of the eyestalk part, and retain the outer shell of the cornea part; Step 2: Fixative selection: Fix at room temperature with 4 - 6% paraformaldehyde for 25 - 35 minutes; Step 3: Dehydration treatment; Step 4: Embed the tissue block after infiltration with modified paraffin; Step 5: Use a microtome to continuously section the sample, with a section thickness of 4 μm; Step 6: Flatten the sections in a 37°C water bath and make them lie flat on a polylysine-treated glass slide; Step 7: After incubating at 37°C for 6 - 12 hours, dewax and rehydrate; Step 8: After staining the sample, use a 50% sterile glycerol aqueous solution to cover-slip and preserve the sample; The specific operating steps of the dehydration treatment are as follows: S01: Add 2 - 5% carbon nanotube modifier to the tissue sample in Step 3; The preparation method of the carbon nanotube modifier is as follows: S11: Send the carbon nanotubes into a hydrochloric acid solution 5 - 10 times their amount, stir evenly, and then add 5 - 10% of the silane coupling agent KH560 and 1 - 4% of the sodium alginate solution based on the total amount of carbon nanotubes; S12: Add the graphene additive to the product of S11 according to a weight ratio of 1:6, stir thoroughly, and finally wash with water and dry to obtain the carbon nanotube modifier; S02: Subsequently, dehydrate with 30% ethanol for 60 minutes, 50% ethanol for 60 minutes, 70% ethanol for 60 minutes, 95% ethanol for 60 minutes, and dehydrate with absolute ethanol twice, 30 minutes each time; Then dehydrate with xylene and absolute ethanol in a ratio of 1:1 for 5 minutes and dehydrate with xylene twice, 3 minutes each time; The preparation method of the graphene additive is: Add graphene to a sodium dodecyl sulfate solution with a mass fraction of 5 - 10% 2 - 5 times its amount, then add 1 - 5% lanthanum sulfate and 1 - 4% 4-aminophenylboronic acid, and continue to stir thoroughly to obtain the graphene additive.

2. The preparation method of the paraffin section of the mantis shrimp eyeball according to claim 1, characterized in that, The mass fractions of the hydrochloric acid solution and the sodium alginate solution are 2 - 5% and 10 - 15% respectively.

3. The preparation method of a paraffin section of a mantis shrimp eyeball according to claim 1, wherein, In the preparation of the graphene additive, the stirring speed for thorough stirring is 1000 - 1500 r / min, the stirring time is 20 - 30 minutes, and the stirring temperature is 45 - 55°C.

4. The preparation method of a paraffin section of a mantis shrimp eyeball according to claim 1, wherein, The preparation method of the modified paraffin is as follows: S01: Send bentonite into 3 - 5 times deionized water and stir to disperse it thoroughly, and finally wash with water and dry; S02: Heat and cool the bentonite in S01, and set it aside after the treatment; S03: Add 5 - 10 parts of chitosan and 1 - 4 parts of sodium alginate to 10 - 15 parts of deionized water, then add 1 - 4 parts of sodium carboxymethylcellulose and 1 - 3 parts of yttrium nitrate, and stir evenly to obtain a modified solution; S04: Stir the bentonite treated in S02 and the modified solution in S03 evenly according to a weight ratio of 1:5, wash with water and dry, and finally mix and prepare with paraffin according to a weight ratio of 1:7 to obtain the modified paraffin.

5. A method for preparing paraffin sections of mantis shrimp eyeballs according to claim 4, characterized in that The specific steps of the heat and cold treatment of bentonite are as follows: First, heat the bentonite to 400 - 450 °C, keep it warm for 10 - 20 min, then cool it to 150 - 160 °C at a rate of 3 - 5 °C / min, keep it warm for 5 - 10 min, and finally cool it with water to room temperature. That's all.

6. The preparation method of the paraffin section of the mantis shrimp eyeball according to claim 5, characterized in that, The temperature of the water cooling is 3 - 6 °C.

7. A method for preparing paraffin sections of mantis shrimp eyes, according to claim 1, characterized in that, The specific operation steps of dewaxing and rehydration in step seven are as follows: treat with xylene for 10 min, treat with a mixture of xylene and absolute ethanol at a ratio of 1:1 for 3 min, treat with 95% ethanol for 3 min, treat with 80% ethanol for 3 min, treat with 70% ethanol for 3 min, treat with 50% ethanol for 3 min, treat with 30% ethanol for 3 min, and treat with distilled water for 3 min.

8. A method for preparing paraffin sections of mantis shrimp eyes, according to claim 7, characterized in that, For the staining, hematoxylin - eosin dye is used to stain the sample.

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