CN105241685A - Preparation method of skin microscopic tissue slice of hynobiidae animals - Google Patents
Preparation method of skin microscopic tissue slice of hynobiidae animals Download PDFInfo
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- CN105241685A CN105241685A CN201510487750.0A CN201510487750A CN105241685A CN 105241685 A CN105241685 A CN 105241685A CN 201510487750 A CN201510487750 A CN 201510487750A CN 105241685 A CN105241685 A CN 105241685A
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Abstract
The invention discloses a preparation method of a skin microscopic tissue slice of hynobiidae animals and belongs to the technical field of microscopic tissue slices. The preparation method includes following steps: 1) dehydration: successively soaking skin tissue, which has been subjected to fixing treatment, of the hynobiidae animals into ethanol solutions being 50%, 70%, 80%, 90%, 95% and 100% in concentration gradient to dehydrate the skin tissue, wherein dehydration time in each concentration gradient is 45-60 min; (2) transparent treatment: soaking the dehydrated skin tissue into a mixed liquid of 100% ethanol and dimethylbenzene for 2-3 min, and then soaking the dehydrated skin tissue into dimethylbenzene for 10-30 min; (3) wax soaking: soaking the skin tissue being subjected to the transparent treatment into a mixed liquid of dimenthylbenzene and paraffin for 20-30 min, and then soaking the skin tissue into paraffin respectively by two times, each time lasts for 45-60 min; and (4) embedding the wax-soaked skin tissue, and performing slice cutting, slice expansion, slice pasting, slice baking, dyeing and seal-fixation to obtain the skin microscopic tissue slice. The preparation method is simple in process and convenient to operate. The prepared slice is complete and shows a clear image structure.
Description
Technical field
The present invention relates to a kind of preparation method of amphibian skin microscopic tissue sections, be specifically related to the preparation method cut into slices for the Hynobiidae animal skin tissue of microexamination, belong to microscopic tissue sections technical field.
Background technology
Skin is the first natural cover for defense of amphibian body, in the process adapted to and evolve, serve as breathing, regulated water balance, ion transportation, opposing hunted many-sided functions such as trencherman, antibacterium, fungi, the complicacy of these function embodiments skin molds feature and the potential physiological property of skin texture, play an important role to maintaining in the wide habitat of the existence of amphibian, developing and adaptation and various ecologic environment.
Hynobiidae animal is the distinctive Urodela amphibian in Asia, is also the third-largest section in existing raw Caudata 10 section, existing known 66 kinds of the whole world, is subordinate to 2 subfamilies, 10 and belongs to.Existing known 2 subfamilies 8 of China belong to 30 kinds, are mainly distributed in northeast, Southwest Mountainous Areas, Central China and Taiwan.Hynobiidae animal living environment can be divided into aquatic type and terrestrial type, due to the difference of skin molds architectural feature between the Hynobiidae animal that living environment causes different living environment, the difference of these morphological features is that the adaptive evolution research of Hynobiidae animal provides the foundation data; Hynobiidae animal is considered to monoid the most original in Urodela simultaneously, therefore the Study on Evolution that the research of skin molds structure can be amphibian provides foundation.Therefore skin molds architectural feature is in amphibian, the research particularly in Hynobiidae animal receives the concern of numerous scholar.
Study the Morphologic Characteristics of skin, the making of skin paraffin section is absolutely necessary a step.Non-patent literature " Senile Mouse of Hynobiidae animal formalness characteristic sum skin histology " (to, 2014) a kind of Chinese salamander skin paraffin section method for making is disclosed, comprise: (1) draws materials: the Chinese salamander skin histology block gathered is generally unsuitable excessive, be advisable with (3 ~ 5mm) × (3 ~ 5mm), running water 24h after fixing; (2) dewater: put into 70% ethanol 2h, 80% ethanol 2h, 85% ethanol 1h, 90% ethanol 1h, 95% ethanol 1h, 100% ethanol I 10min, 100% ethanol II 10min successively; (3) transparent: the tissue after dehydration is put into normal butyl alcohol I 15min, and normal butyl alcohol II 15min carries out transparent processing; (4) waxdip: skin histology is put into paraffin I waxdip 30min, then put into paraffin refined wax II waxdip 2h; (5) embed: the paraffin embedding using about 75 DEG C; (6) cut into slices: first repair parallel by wax stone surrounding with single-edge blade and be cut into square or rectangle, wax stone is sticked on wooden unit, and adjustment blade about becomes 5 ° of angles with paraffin section, and slice thickness is 4 ~ 6 μm; (7) open up sheet, paster, drying: rolled by the tissue writing brush cut, slowly divide and extend piece in the warm water of 44 DEG C, then section provoked with microslide, put into baking box, dry sheet and be as the criterion so that slide is obviously not water stain; (8) HE dyeing: paraffin section tissue is put into dimethylbenzene 15min, 100% ethanol 10min, 95% ethanol 5min, 85% ethanol 5min, 75% ethanol 5min, distilled water 5min, haematoxylin 10min, running water 5min, 1% hydrochloride alcohol color separation about 40s, running water oil blackeite 30min, 70% ethanol 2min, 80% ethanol 2min, Yihong ethanol 5min, 95% ethanol 4min, 100% ethanol 4min, put into dimethylbenzene 10min (amounting to 1h58min) again, then use neutral gum mounting; (9) sealing: neutral gum is dripped at place at microslide 1/3, the right side that cover glass clamped lightly by thin tweezer is held with the right hand, and its left side is placed on the left side of natural gum, then left hand holds the left side that dissecting needle buttresses cover glass, and tweezers unclamp and decline gradually by the right hand, slowly extract out, make natural gum cover glass launch equably and will wherein bubble discharge, drying, wipes clean the natural gum of unnecessary spilling around cover glass, to obtain final product with dimethylbenzene.But the method having the following disadvantages: (1) dehydration not exclusively, causes paraffin infiltration incomplete, causes tissue to occur broken or empty when cutting into slices, cutting into slices during the sheet of stand and scattering, supporting spring and painted bad etc. during dyeing; (2) normal butyl alcohol dehydrating effect is not good; (3) waxdip process is owing to lacking transition step, and cause the dimethylbenzene carried from transparent step to enter paraffin, thus cause paraffin concentration to reduce, waxdip overlong time causes tissue block firmly crisp in addition.
Summary of the invention
The object of this invention is to provide a kind of preparation method of Hynobiidae animal skin microscopic tissue sections, the method effectively can remove the moisture in tissue block, prevents paraffin infiltration incomplete; Improve transparent effect, the waxdip time shortens relatively, and tissue block can be prevented firmly crisp.
In order to realize above object, the technical solution adopted in the present invention is:
The preparation method of Hynobiidae animal skin microscopic tissue sections, comprises the following steps:
(1) dewater: the Hynobiidae animal skin tissue through fixing process is immersed in the ethanol of concentration gradient 50%, 70%, 80%, 90%, 95%, 100% successively and dewaters, wherein 95%, 100% ethanol respectively repeats twice, each serial dehydration 45 ~ 60min;
(2) transparent: the tissue through skin treatment of dewatering to be immersed 2 ~ 3min in the mixed liquor of 100% ethanol and dimethylbenzene, after taking-up, immerses 10 ~ 30min in dimethylbenzene again;
(3) waxdip: the mixed liquor 20 ~ 30min skin histology through transparent processing being put into dimethylbenzene and paraffin, puts into paraffin after taking-up, each 45 ~ 60min more at twice;
(4) skin histology through waxdip process is embedded, successively through section, exhibition sheet, paster, roasting sheet, dyeing, sealing process, to obtain final product.
In step (1), the increase of concentration of alcohol and the increase of dewatering time more effectively can remove the moisture in tissue block, thus ensure that paraffin penetrates into histocyte fully, otherwise paraffin infiltration will be caused incomplete, tissue is caused to occur when cutting into slices broken or empty, during the sheet of stand, section is scattered, supporting spring and painted bad etc. during dyeing.
Dewater described in step (1) and at room temperature carry out.
Described in step (2), the volume ratio of 100% ethanol and dimethylbenzene is 1:1.
In step (2), the transparent effect of dimethylbenzene in the section of Hynobiidae animal skin tissue is best, can dissolve paraffin preferably.
Adopt in step (3) object of the mixed liquor of dimethylbenzene and paraffin to be increase a transition step between transparent and waxdip step, bring too much dimethylbenzene in transparent step enter paraffin in case cut into slices, thus cause the reduction of paraffin concentration; Can suitably shorten the waxdip time, in case waxdip overlong time causes tissue block firmly crisp after increasing the transition step of dimethylbenzene and paraffin simultaneously.
Described in step (3), the volume ratio of dimethylbenzene and paraffin is 1:1.
Described in step (4), the thickness of section is 4 ~ 6 μm, and section is placed in the water of 42 ~ 45 DEG C opens up sheet.
Paster described in step (4), roasting sheet are: slice takes out after naturally stretching and putting down from water, control solid carbon dioxide divides, dry 2 ~ 4h at being placed in 37 ~ 45 DEG C, or natural drying one night under room temperature, also maybe section is placed on microslide, hold microslide after adding a small amount of water to heat on spirit lamp, section is stretched.
Dyeing described in step (4) is H.E dyeing, operation steps is: section is placed in successively dimethylbenzene I 10min, dimethylbenzene II 10min, 100% ethanol I 5min, 100% ethanol II 5min, 95% ethanol 5min, 85% ethanol 5min, 75% ethanol 5min, water 5min, haematine dye liquor 10 ~ 20min, running water 5min, be placed in HCl-ethanol differentiation liquid 20 ~ 40s again, running water 10 ~ 30min, microscopy, running water 2min, be placed in 70% ethanol 1min more successively, 85% ethanol 1min, eosin stain 2min, 95% ethanol I 2min, 95% ethanol II 2min, 100% ethanol I 2min, 100% ethanol II 2min, dimethylbenzene I 5min, dimethylbenzene II 5min.Wherein, I, II only represents number of processes, and process 10min as dimethylbenzene I 10min, dimethylbenzene II 10min are respectively dimethylbenzene first time, dimethylbenzene second time processes 10min.
Described haematine dye liquor is: be dissolved in by 1g haematine in 20mL100% ethanol and obtain first liquid, and 50g potassium alum is dissolved in 300mL distilled water and obtains second liquid; By first liquid, the mixing of second liquid, boil 2 ~ 3min, then adding distil water is settled to 1000mL; Finally add 0.2g sodium iodate, to obtain final product.
Described eosin stain is: joined in 0.5g Yihong in 100mL85% ethanol, dissolves completely, to obtain final product.
Described HCl-ethanol differentiation liquid is: joined by 1mL hydrochloric acid in 99mL70% ethanol, mixing, to obtain final product.
Beneficial effect of the present invention:
In the present invention the preparation method of Hynobiidae animal skin microscopic tissue sections comprise draw materials, fix, rinse, dewater, transparent, waxdip, embedding, the step such as finishing, section and exhibition sheet, paster and roasting sheet, H.E dye, sealing, flow process is simple, easy to operate.Meanwhile, the method effectively can remove the moisture in tissue block, prevents paraffin infiltration incomplete; Improve transparent effect, the waxdip time shortens relatively, and tissue block can be prevented firmly crisp, the section obtained is complete, picture structure is clear, for research Hynobiidae animal skin structure provides technical support, is applicable to the research of laboratory study worker to Hynobiidae animal skin tissue morphosis.
Accompanying drawing explanation
Fig. 1 is Chinese Chinese salamander (Hynobiuschinesis) skin paraffin section micrograph in embodiment 1.
Embodiment
Following embodiment is only described in further detail the present invention, but does not form any limitation of the invention.
Embodiment 1
The preparation of dye liquor and differentiation liquid:
Haematine dye liquor: 1g haematine is dissolved in 20mL100% ethanol and obtains first liquid, 50g potassium alum is dissolved in 300mL distilled water and obtains second liquid; By first liquid, the mixing of second liquid, boil 2 ~ 3min, then adding distil water is settled to 1000mL; Finally add 0.2g sodium iodate, to obtain final product.
Eosin stain: joined in 0.5g Yihong in 100mL85% ethanol, dissolves completely, to obtain final product.If will not dye, the glacial acetic acid dilution of 0.2% can be added, to promote eosin stains.
HCl-ethanol differentiation liquid: joined by 1mL hydrochloric acid in 99mL70% ethanol, mixing, to obtain final product.
The preparation method of Hynobiidae animal China Chinese salamander (Hynobiuschinesis) skin microscopic tissue sections in the present embodiment, comprises the following steps:
(1) draw materials: at room temperature, use tweezers to be picked up by Hynobiidae back part of animal skin, then use skin needed for scissors clip, put into 10% formalin room temperature preservation, the skin histology block size gathered is 4mm × 4mm, and tangent plane should be point-blank;
(2) fixing: to be dropped into by the skin histology of collection in 10% formalin immobile liquid (or 75% alcohol fixation liquid) and be fixed, consumption is 15 times (10 ~ 20 by) of tissue block volume, and carries out mark, is convenient to identify;
(3) rinse: the skin histology water after fixing is rinsed 8min (5 ~ 10min);
(4) dewater: at room temperature dewatering agent (ethanol) is made into different gradient concentrations with distilled water, then skin histology is inserted, dewater step by step, namely dewater in the ethanol of 50%, 70%, 80%, 90%, 95%, 100% successively, wherein 95%, 100% ethanol respectively repeats twice, the time 50min of ethanol dehydration at different levels; If adopt 75% alcohol fixation liquid-solid fixed in step (3), then directly add in 80% alcohol and carry out dewater (as spent the night, tissue block should rest in 70% ethanol);
(5) transparent: in two steps transparent, the first step adopts the transparent liquid of 100% ethanol and dimethylbenzene 1:1 preparation by volume, and clearing time is 2.5min, and second step adopts pure transparent solution of xylene, and clearing time is 20min;
(6) waxdip: transparent tissue is put into the dimethylbenzene of volume ratio 1:1 and the mixed liquor 25min of paraffin, and then put into the container filling 58 DEG C of pure waxes at twice, each waxdip 50min, the saturating wax of 100min is complete altogether;
(7) embed: the paraffin injecting thawing in embedded box, the tissue block of waxdip is inserted wherein (make the tissue surface for cutting downward), wax stone is cooled rapidly hard solid, embedded tissue block can be preserved for a long time in wax stone;
(8) trimming of paraffin mass: paraffin mass on embedded box is done suitable trimming, to save blade;
(9) section and exhibition sheet: slice thickness 5 μm, has cuting slice to be placed in 42 DEG C of warm water and open up sheet;
(10) paster and roasting sheet: treat paraffin section naturally stretch flat after fish for from water lightly, position of setting right, control solid carbon dioxide divides, and then puts into 40 DEG C of dry 3h of incubator;
H.E staining (11) : H.E staining, according to the following process to dye the slice in xylene in turn Ⅰ (10 min) - xylene Ⅱ (10 min) and 100% alcohol Ⅰ Ⅱ (5 min) and 100% alcohol (5 min) to 95% alcohol (5 min), 85% alcohol (5 min) and 75% alcohol (5 min) to distilled water immersion (5 min), hematoxylin dyeing liquid (15 min) - water flushing (5 min) - HCl - liquid alcohol differentiation (30 s) - water flushing (min) 20 - distilled water rinse after microscopy (2 min) to 70% alcohol (1 min), 85% alcohol (1 min) and eosin dye solution (2 min) - > 95% alcohol Ⅰ Ⅱ (2 min) - > 95% alcohol (2 min) - > 100% alcohol Ⅰ Ⅱ (2 min) - > 100% alcohol (2 min) - xylene Ⅰ (5 min) - xylene Ⅱ (5 min);
(12) sealing: skin histology edge after dyeing drips neutral gum, the right side that cover glass clamped lightly by thin tweezer is held with the right hand, and its left side is placed on the left side of natural gum, then left hand holds the left side that dissecting needle buttresses cover glass, tweezers unclamp and decline gradually by the right hand, slowly extract out, such natural gum launches equably at cover glass and wherein bubble is discharged, the natural gum overflow on cover glass Zhou Duoyu limit is wiped clean with dimethylbenzene, section placed steadily, weight a little, treats that gummy drying is solidified, obtain, section can be preserved for a long time.
In the present embodiment, as shown in Figure 1, in figure, Ep is epidermal area to Hynobiidae animal China Chinese salamander (Hynobiuschinesis) skin paraffin section micrograph, and SC is fine and close skin corium, and P is chromatophore, and M is muccus gland, and CV is capillary.
Points for attention:
1, processed uses the effect of gradient concentration ethanol to be utilize the ethanol of variable concentrations progressively to slough moisture in histocyte in step (4), and be unlikely to make histocyte deformation effect observing effect, strictly the time should be held, in order to avoid dehydration excessively when it should be noted that and adopt high concentration ethanol dehydration.
2, the dimethylbenzene that in step (5), transparent processing adopts has the effect of hyaline tissue, utilize dimethylbenzene to occupy ethanol position in cell thus reach the effect of hyaline tissue, it should be noted that the moment should note transparency and transparent suitably after put into paraffin immediately.The time stopped in should noting every grade during dehydration determines according to the character of the size of material, character and immobile liquid.
3, step (5) if in tissue block have local when can not transparently namely have in white casse state, be then because of dehydration thoroughly caused by, should return and again dewater, and then transparent.
4, in step (6), the effect of waxdip is the characteristic utilizing paraffin and dimethylbenzene to dissolve each other, and makes paraffin occupy the position of dimethylbenzene in tissue, thus is conducive to section.
5, its effect of the dimethylbenzene that in step (11), dyeing course uses is dewaxing, haematoxylin dyeing nucleus, eosin stains tenuigenin, it should be noted that the dyeing course palpus strict assurance time is in order to avoid fall short of success for lack of final effort.
In other embodiments of the invention, the value relating to parameter in each step can make the appropriate adjustments, as in step (4) processed, the ethanol dehydration time at different levels suitably can be chosen at 45 ~ 60min, process 60min when tissue block is larger, process 45min when tissue block is less, for another example in step (5) transparent processing, the time that second step is transparent suitably can be chosen at 10 ~ 30min, can stop when reaching transparent effect.In given scope, suitably each parameter value of adjustment is this area routine techniques, no longer illustrates herein.
Claims (9)
1. the preparation method of Hynobiidae animal skin microscopic tissue sections, is characterized in that: comprise the following steps:
(1) dewater: the Hynobiidae animal skin tissue through fixing process is immersed in the ethanol of concentration gradient 50%, 70%, 80%, 90%, 95%, 100% successively and dewaters, wherein 95%, 100% ethanol respectively repeats twice, each serial dehydration 45 ~ 60min;
(2) transparent: the skin histology through processed to be immersed 2 ~ 3min in the mixed liquor of 100% ethanol and dimethylbenzene, immerse 10 ~ 30min in dimethylbenzene again after taking-up;
(3) waxdip: the mixed liquor 20 ~ 30min skin histology through transparent processing being put into dimethylbenzene and paraffin, puts into paraffin after taking-up, each 45 ~ 60min more at twice;
(4) skin histology through waxdip process is embedded, successively through section, exhibition sheet, paster, roasting sheet, dyeing, sealing process, to obtain final product.
2. preparation method according to claim 1, is characterized in that: described in step (2), the volume ratio of 100% ethanol and dimethylbenzene is 1:1.
3. preparation method according to claim 1, is characterized in that: described in step (3), the volume ratio of dimethylbenzene and paraffin is 1:1.
4. preparation method according to claim 1, is characterized in that: described in step (4), the thickness of section is 4 ~ 6 μm, and section is placed in the water of 42 ~ 45 DEG C opens up sheet.
5. preparation method according to claim 1, it is characterized in that: paster described in step (4), roasting sheet are: slice takes out after naturally stretching and putting down from water, control solid carbon dioxide divides, dry 2 ~ 4h at being placed in 37 ~ 45 DEG C, or under room temperature natural drying one night.
6. preparation method according to claim 1, it is characterized in that: dyeing described in step (4) is H.E dyeing, operation steps is: section is placed in successively dimethylbenzene I 10min, dimethylbenzene II 10min, 100% ethanol I 5min, 100% ethanol II 5min, 95% ethanol 5min, 85% ethanol 5min, 75% ethanol 5min, water 5min, haematine dye liquor 10 ~ 20min, running water 5min, be placed in HCl-ethanol differentiation liquid 20 ~ 40s again, running water 10 ~ 30min, microscopy, running water 2min, be placed in 70% ethanol 1min more successively, 85% ethanol 1min, eosin stain 2min, 95% ethanol I 2min, 95% ethanol II 2min, 100% ethanol I 2min, 100% ethanol II 2min, dimethylbenzene I 5min, dimethylbenzene II 5min.
7. preparation method according to claim 6, is characterized in that: described haematine dye liquor is: be dissolved in by 1g haematine in 20mL100% ethanol and obtain first liquid, and 50g potassium alum is dissolved in 300mL distilled water and obtains second liquid; By first liquid, the mixing of second liquid, boil 2 ~ 3min, then adding distil water is settled to 1000mL; Finally add 0.2g sodium iodate, to obtain final product.
8. preparation method according to claim 6, is characterized in that: described eosin stain is: joined in 0.5g Yihong in 100mL85% ethanol, dissolves completely, to obtain final product.
9. preparation method according to claim 6, is characterized in that: described HCl-ethanol differentiation liquid is: joined by 1mL hydrochloric acid in 99mL70% ethanol, mixing, to obtain final product.
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Cited By (6)
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| CN107843470A (en) * | 2017-10-13 | 2018-03-27 | 济南金域医学检验中心有限公司 | A kind of preparation method of Skin slice |
| CN109115585A (en) * | 2018-08-23 | 2019-01-01 | 钱金林 | A kind of Automatic tissue slice overflow dyeing machine that dyeing quality can be improved and its method |
| CN110146356A (en) * | 2019-05-22 | 2019-08-20 | 山西医科大学 | A kind of experiment mice adipose tissue paraffin section tissue treatment methods |
| CN111238900A (en) * | 2020-02-28 | 2020-06-05 | 青海民族大学 | Preparation method of plateau wood frog skin paraffin section |
| CN112067380A (en) * | 2020-08-06 | 2020-12-11 | 佛山科学技术学院 | Improved mouse bone marrow dehydration method |
| CN114689407A (en) * | 2022-04-14 | 2022-07-01 | 四川大学华西医院 | Method for manufacturing paraffin section of animal microtissue |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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| CN109115585A (en) * | 2018-08-23 | 2019-01-01 | 钱金林 | A kind of Automatic tissue slice overflow dyeing machine that dyeing quality can be improved and its method |
| CN110146356A (en) * | 2019-05-22 | 2019-08-20 | 山西医科大学 | A kind of experiment mice adipose tissue paraffin section tissue treatment methods |
| CN111238900A (en) * | 2020-02-28 | 2020-06-05 | 青海民族大学 | Preparation method of plateau wood frog skin paraffin section |
| CN112067380A (en) * | 2020-08-06 | 2020-12-11 | 佛山科学技术学院 | Improved mouse bone marrow dehydration method |
| CN114689407A (en) * | 2022-04-14 | 2022-07-01 | 四川大学华西医院 | Method for manufacturing paraffin section of animal microtissue |
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| CN105241685B (en) | 2018-04-13 |
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