CN105241685B - The preparation method of Hynobiidae animal skin microscopic tissue sections - Google Patents
The preparation method of Hynobiidae animal skin microscopic tissue sections Download PDFInfo
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Abstract
The invention discloses a kind of preparation method of Hynobiidae animal skin microscopic tissue sections, belong to microscopic tissue sections technical field.This method comprises the following steps：(1) it is dehydrated：Hynobiidae animal skin tissue through fixing process is immersed in the ethanol of concentration gradient 50%, 70%, 80%, 90%, 95%, 100% successively and is dehydrated, each 45~60min of serial dehydration；(2) it is transparent：Skin histology through dehydration is immersed to 2~3min in the mixed liquor of 100% ethanol and dimethylbenzene, immerses 10~30min in dimethylbenzene after taking-up again；(3) waxdip：Skin histology through transparent processing is put into 20~30min in the mixed liquor of dimethylbenzene and paraffin, is put at twice in paraffin again after taking-up, every time 45~60min；(4) skin histology handled through waxdip is embedded, sliced successively, exhibition piece, patch, roasting piece, dyeing, sealing processing, to obtain the final product；Its flow is simple, easy to operate, and the section of acquisition is complete, and picture structure is clear.
Description
Technical field
The present invention relates to a kind of preparation method of amphibian skin microscopic tissue sections, and in particular to for microexamination
Hynobiidae animal skin tissue section preparation method, belong to microscopic tissue sections technical field.
Background technology
Skin is first of natural cover for defense of amphibian body, during adaptation and evolution, has served as breathing, has adjusted
Water-saving balance, ion transport, resistance hunt many functions such as trencherman, antibacterium, fungi, these function embodiments skin molds
The complexity and the potential physiological property of skin texture of feature are learned, the existence, developing and adaptation to maintenance amphibian are wide
Habitat and various ecological environment in play an important role.
Hynobiidae animal is the distinctive Urodela amphibian in Asia, and now the third-largest section in raw 10 section of Caudata, entirely
The world has known 66 kinds, is subordinate to 2 subfamilies, 10 categories.China known 2 subfamilies 8 belong to 30 kinds, be distributed mainly on northeast, Southwest Mountainous Areas,
Central China and Taiwan.Hynobiidae animal living environment can be divided into aquatic type and terrestrial type, since living environment causes different lifes
The difference of skin molds architectural feature between the Hynobiidae animal of environment living, the difference of these morphological features is Hynobiidae animal
The data that provide the foundation are studied in adaptive evolution；Hynobiidae animal is considered as the monoid of most original in Urodela at the same time, therefore skin
The research of morphosis can provide foundation for the Study on Evolution of amphibian.Therefore skin molds architectural feature is in amphibian,
Research particularly in Hynobiidae animal is paid close attention to be subject to numerous scholars.
The Morphologic Characteristics of skin are studied, the making of skin paraffin section is an essential step.Non- patent text
Offer《The Senile Mouse of Hynobiidae animal formalness feature and skin histology》(opening to 2014) discloses a kind of Chinese salamander skin
Paraffin section production method, including：(1) draw materials：The Chinese salamander skin histology block gathered generally should not be too large, with (3~5mm) ×
(3~5mm) is advisable, and flowing water rinses 24h after fixing；(2) it is dehydrated：It is sequentially placed into 70% ethanol 2h, 80% ethanol 2h, 85% ethanol
1h, 90% ethanol 1h, 95% ethanol 1h, 100% ethanol, I 10min, 100% ethanol, II 10min；(3) it is transparent：Will be dewatered
Tissue is put into I 15min of n-butanol, and II 15min of n-butanol carries out transparent processing；(4) waxdip：Skin histology is put into paraffin I to soak
Wax 30min, places into II waxdip 2h of paraffin refined wax；(5) embed：Use 75 DEG C or so of paraffin embedding；(6) cut into slices：With single side knife
Wax stone surrounding is first repaired parallel and is cut into square or rectangle by piece, and wax stone is adhered on wooden unit, and adjustment blade is cut with paraffin
For agreement that contracts a film or TV play to an actor or actress into 5 ° of angles, slice thickness is 4~6 μm；(7) piece, patch, drying are opened up：The tissue cut is rolled with writing brush, is slowly put down
Spread out and extended piece in 44 DEG C of warm water, then section is provoked with glass slide, is put into baking box, it is not bright with slide to dry piece
Subject to showing water stain；(8) HE is dyed：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, flowing water rinse 5min, 1% hydrochloric acid wine
Smart color separation 40s or so, flowing water rinse oil blackeite 30min, 70% ethanol 2min, 80% ethanol 2min, Yihong ethanol 5min, 95% second
Alcohol 4min, 100% ethanol 4min, place into dimethylbenzene 10min (amounting to 1h58min), then with neutral gum mounting；(9) seal
Gu：Neutral gum is dripped at glass slide 1/3, holds the right side that thin tweezer lightly clamps coverslip with the right hand, and its left side is put
On the left side of natural gum, then left hand holds the left side that dissecting needle buttresses coverslip, and the right hand, which unclamps tweezers, to be gradually reduced, and is slowly taken out
Go out, natural gum is unrolled evenly and will wherein bubble be discharged in coverslip, dry, it is unnecessary around coverslip to be wiped clean with dimethylbenzene
The natural gum of spilling, to obtain the final product.However, this method has the following disadvantages：(1) dehydration is incomplete, causes paraffin infiltration incomplete,
Cause tissue broken or cavity occur in section, when spreading out piece section scatter, supporting spring and colour bad etc. during dyeing；(2) positive fourth
Dehydration of alcohols is ineffective；(3) waxdip process causes to enter paraffin from the dimethylbenzene that transparent step carries due to lacking transition step
In, so as to cause the reduction of paraffin concentration, in addition waxdip overlong time causes tissue block brittle.
The content of the invention
The object of the present invention is to provide a kind of preparation method of Hynobiidae animal skin microscopic tissue sections, this method can have
Effect removes tissue moisture in the block, prevents that paraffin infiltration is not complete；Transparent effect is improved, the opposite shortening of waxdip time, can prevent group
It is brittle to knit block.
In order to realize the 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) it is dehydrated：By the Hynobiidae animal skin tissue through fixing process immerse successively concentration gradient 50%, 70%,
80%th, it is dehydrated in 90%, 95%, 100% ethanol, wherein 95%, 100% ethanol is respectively repeated twice, each serial dehydration 45
~60min；
(2) it is transparent：Tissue through dehydration skin treatment is immersed to 2~3min in the mixed liquor of 100% ethanol and dimethylbenzene,
10~30min in dimethylbenzene is immersed after taking-up again；
(3) waxdip：Skin histology through transparent processing is put into 20~30min in the mixed liquor of dimethylbenzene and paraffin, is taken
It is put at twice in paraffin again after going out, every time 45~60min；
(4) skin histology handled through waxdip is embedded, sliced successively, exhibition piece, patch, roasting piece, dyeing, at sealing
Reason, to obtain the final product.
The increase of concentration of alcohol and the increase of dewatering time being capable of significantly more efficient removal tissue water in the block in step (1)
Point, so as to ensure that paraffin sufficiently penetrates into histocyte, paraffin infiltration otherwise will be caused incomplete, and cause group in section
Weave and now crush or cavity, when spreading out piece section scatter, supporting spring and colour bad etc. during dyeing.
It is dehydrated described in step (1) and carries out at room temperature.
The volume ratio of 100% ethanol and dimethylbenzene described in step (2) is 1:1.
Transparent effect of the dimethylbenzene in Hynobiidae animal skin tissue cuts into slices is best in step (2), can preferably dissolve
Paraffin.
The purpose that the mixed liquor of dimethylbenzene and paraffin is used in step (3) is to increase by one between the transparent step with waxdip
Transition step, brings excessive dimethylbenzene into transparent step to prevent section and enters paraffin, so as to cause the reduction of paraffin concentration；Together
It can suitably shorten the waxdip time after the transition step of Shi Zengjia dimethylbenzene and paraffin, cause tissue block crisp to prevent waxdip overlong time
Firmly.
The volume ratio of dimethylbenzene and paraffin described in step (3) is 1:1.
The thickness cut into slices described in step (4) is 4~6 μm, and section, which is placed in 42~45 DEG C of water, opens up piece.
Patch described in step (4), roasting piece are：Slice stretch naturally it is flat after taken out from water, it is drained, be placed in 37
Dry 2~4h at~45 DEG C, or a night is spontaneously dried at room temperature, also or by section it is placed on glass slide, after adding a small amount of water
Hold glass slide to heat on alcolhol burner, stretch section.
Dyeing is dyed for H.E described in step (4), and operating procedure is：Section is sequentially placed into I 10min of dimethylbenzene, diformazan
II 10min of benzene, 100% ethanol, I 5min, 100% ethanol, II 5min, 95% ethanol 5min, 85% ethanol 5min, 75% ethanol
5min, water 5min, hematoxylin 10~20min of dye liquor, flowing water rinse 5min, then are placed in HCl- ethanol differentiation 20~40s of liquid, flowing water
10~30min is rinsed, microscopy, flowing water rinses 2min, then is sequentially placed into 70% ethanol 1min, 85% ethanol 1min, eosin stain
2min, 95% ethanol, I 2min, 95% ethanol, II 2min, 100% ethanol, I 2min, 100% ethanol, II 2min, I 5min of dimethylbenzene,
II 5min of dimethylbenzene.Wherein, I, II number of processes is only represented, if I 10min of dimethylbenzene, II 10min of dimethylbenzene are respectively dimethylbenzene
Processing 10min, dimethylbenzene handle 10min for the second time for the first time.
The hematoxylin dye liquor is：1g hematoxylins are dissolved in 100% ethanol of 20mL and obtain solution A, 50g potassium alums are dissolved in
Second liquid is obtained in 300mL distilled water；Solution A, second liquid are mixed, boil 2~3min, then add distilled water to be settled to 1000mL；Finally plus
Enter 0.2g sodium iodates, to obtain the final product.
The eosin stain is：0.5g Yihong is added in 85% ethanol of 100mL, dissolving is complete, to obtain the final product.
The HCl- ethanol breaks up liquid：1mL hydrochloric acid is added in 70% ethanol of 99mL, is mixed, to obtain the final product.
Beneficial effects of the present invention：
In the present invention preparation method of Hynobiidae animal skin microscopic tissue sections include materials, it is fixed, rinse, dehydration,
Transparent, waxdip, embedding, finishing, section and exhibition piece, patch and roasting piece, H.E dyeing, sealing, flow is simple, operation side
Just.Meanwhile this method can effectively remove tissue moisture in the block, prevent that paraffin infiltration is not complete；Improve transparent effect, waxdip time
It is opposite to shorten, it can prevent that tissue block is brittle, the section of acquisition is complete, and picture structure is clear, to study Hynobiidae animal skin knot
Structure provides technical support, suitable for research of the laboratory research worker to Hynobiidae animal skin tissue morphosis.
Brief description of the drawings
Fig. 1 is Chinese Chinese salamander (Hynobius chinesis) skin paraffin section micrograph in embodiment 1.
Embodiment
Following embodiments are only described in further detail the present invention, but do not form any limitation of the invention.
Embodiment 1
Dye liquor and the preparation for breaking up liquid：
Hematoxylin dye liquor：1g hematoxylins are dissolved in 100% ethanol of 20mL and obtain solution A, 50g potassium alums are dissolved in 300mL steamings
Second liquid is obtained in distilled water；Solution A, second liquid are mixed, boil 2~3min, then add distilled water to be settled to 1000mL；It is eventually adding 0.2g
Sodium iodate, to obtain the final product.
Eosin stain：0.5g Yihong is added in 85% ethanol of 100mL, dissolving is complete, to obtain the final product.If will not dye,
0.2% glacial acetic acid dilution can be added, to promote eosin stains.
HCl- ethanol breaks up liquid：1mL hydrochloric acid is added in 70% ethanol of 99mL, is mixed, to obtain the final product.
The preparation of Hynobiidae animal China Chinese salamander (Hynobius chinesis) skin microscopic tissue sections in the present embodiment
Method, comprises the following steps：
(1) draw materials：At room temperature, Hynobiidae back part of animal skin is picked up using tweezers, then using scissors clip institute
Skin is needed, is put into room temperature preservation in 10% formalin, the skin histology block size of collection is 4mm × 4mm, and section Ying Yi
On bar straight line；
(2) it is fixed：By the skin histology of collection put into 10% formalin fixer (or 75% alcohol fixation liquid) into
Row is fixed, and dosage is 15 times (10~20 by) of tissue block volume, and carries out mark, easy to identify；
(3) rinse：Skin histology water after fixation is rinsed into 8min (5~10min)；
(4) it is dehydrated：Dehydrating agent (ethanol) is made into different gradient concentrations with distilled water at room temperature, then by skin group
Knit and insert, be dehydrated step by step, i.e., be dehydrated successively in 50%, 70%, 80%, 90%, 95%, 100% ethanol, wherein
95%th, 100% ethanol is respectively repeated twice, the time 50min of ethanol dehydrations at different levels；If 75% alcohol fixation is used in step (3)
Liquid is fixed, then is directly added into 80% alcohol and is dehydrated and (need to such as stayed overnight, tissue block should be rested in 70% ethanol)；
(5) it is transparent：It is transparent in two steps, the first step is using 100% ethanol and dimethylbenzene by volume 1:1 prepare it is transparent
Liquid, clearing time 2.5min, second step use pure transparent solution of xylene, clearing time 20min；
(6) waxdip：Transparent tissue is put into volume ratio 1:25min in 1 dimethylbenzene and the mixed liquor of paraffin, then
It is put at twice again in the container for filling 58 DEG C of pure wax, each waxdip 50min, the common saturating waxes of 100min finish；
(7) embed：The paraffin of thawing is injected in embedded box, the tissue block of waxdip, which is inserted, wherein (makes the group to be cut
Surface of second order is downward), wax stone is cooled down rapidly hard solid, embedded tissue block can preserve for a long time in wax stone；
(8) trimming of paraffin mass：Paraffin mass on embedded box is done into appropriate trimming, to save blade；
(9) section and exhibition piece：5 μm of slice thickness, has cuting slice to be placed in 42 DEG C of warm water and opens up piece；
(10) patch and roasting piece：Treat that paraffin section stretches to put down naturally lightly to fish for from water afterwards, position of setting right, drains
Moisture, is then placed in 40 DEG C of incubators dry 3h；
(11) H.E is dyed：H.E dyeing is carried out by below scheme, and section to be contaminated is sequentially placed into dimethylbenzene I (10min) → bis-
I alcohol of (5min) → 100% of toluene II (10min) → 100% alcohol, II alcohol of (5min) → 95% (5min) → 85% alcohol
The alcohol of (5min) → 75% (5min) → distilled water immersion (5min) → hematoxylin dye liquor (15min) → flowing water flushing (5min) →
HCl- alcohol differentiation liquid (30s) → flowing water rinses distilled water flushing (2min) → 70% alcohol (1min) after (20min) → microscopy
→ 85% alcohol (1min) → I alcohol of (2min) → 95% of eosin stain (2min) → 95% alcohol, II wine of (2min) → 100%
II (2min) → dimethylbenzene of smart I alcohol of (2min) → 100%, I (5min) → dimethylbenzene II (5min)；
(12) sealing：Skin histology edge drop neutral gum after dyeing, holds thin tweezer with the right hand and lightly clamps lid glass
The right side of piece, and its left side is placed on the left side of natural gum, then left hand holds the left side that dissecting needle buttresses coverslip, and the right hand is by tweezer
Son, which unclamps, to be gradually reduced, and is slowly extracted out, and so natural gum is unrolled evenly and will wherein bubble discharge in coverslip, is wiped with dimethylbenzene
The natural gum that clean coverslip Zhou Duoyu sides are overflow, section is placed steadily, slightly weight, treats the dry solidification of natural gum, to obtain the final product, section can
It is long-term to preserve.
Hynobiidae animal China Chinese salamander (Hynobius chinesis) skin paraffin section micrograph is as schemed in the present embodiment
Shown in 1, Ep is epidermis in figure, and SC is fine and close skin corium, and P is chromatophore, and M is muccus gland, and CV is capillary.
Points for attention：
1st, dehydration using the effect of gradient concentration ethanol is progressively sloughed using the ethanol of various concentrations in step (4)
Moisture in histocyte, and be unlikely to make histocyte deformation effect observing effect, it should be noted that using high concentration second
Should strictly it be held the time during dehydration of alcohols, in case dehydration is excessive.
2nd, the dimethylbenzene that transparent processing uses in step (5) plays the role of hyaline tissue, is occupied using dimethylbenzene in cell
Ethanol position is so as to reaching the effect of hyaline tissue, it should be noted that should pay attention to constantly transparency and it is transparent it is appropriate after immediately
It is put into paraffin.It should be noted that residence time is depending on the property of the size of material, property and fixer in every grade during dehydration.
3rd, it is because being dehydrated not if when tissue block has part transparent cannot have white cloudy state in step (5)
Caused by thoroughly, it should retract and be dehydrated again, it is then transparent again.
4th, the effect of waxdip is the characteristic dissolved each other using paraffin and dimethylbenzene in step (6), paraffin is occupied two in tissue
The position of toluene, so as to be conducive to cut into slices.
5th, its effect of the dimethylbenzene that dyeing course uses in step (11) is dewaxing, and haematoxylin dyeing nucleus, Yihong contaminates
Cytochrome matter, it should be noted that dyeing course strictly must hold the time in order to avoid falling short of success for lack of final effort.
In other embodiments of the invention, the value of parameter involved in each step can make the appropriate adjustments, as step (4) is de-
In water process, the ethanol dehydration times at different levels can suitably be chosen in 45~60min, handle 60min when tissue block is larger, tissue block compared with
Hour processing 45min, for another example in step (5) transparent processing, the second step transparent time can suitably be chosen in 10~30min, reach
It can stop to during transparent effect.It is this area routine techniques suitably to adjust each parameter value in given scope, herein not
Illustrate again.
Claims (4)
1. the preparation method of Hynobiidae animal skin microscopic tissue sections, it is characterised in that：Comprise the following steps：
（1）Dehydration：By the Hynobiidae animal skin tissue through fixing process immerse successively concentration gradient 50%, 70%, 80%, 90%,
95%th, it is dehydrated in 100% ethanol, wherein 95%, 100% ethanol is respectively repeated twice, each 45 ~ 60min of serial dehydration；
（2）It is transparent：Skin histology through dehydration is immersed to 2 ~ 3min in the mixed liquor of 100% ethanol and dimethylbenzene, after taking-up
10 ~ 30min in dimethylbenzene is immersed again；The volume ratio of 100% ethanol and dimethylbenzene is 1:1；
（3）Waxdip：Skin histology through transparent processing is put into 20 ~ 30min in the mixed liquor of dimethylbenzene and paraffin, after taking-up again
It is put at twice in paraffin, every time 45 ~ 60min；The volume ratio of the dimethylbenzene and paraffin is 1:1；
（4）The skin histology handled through waxdip is embedded, sliced successively, exhibition piece, patch, roasting piece, dyeing, sealing processing, i.e.,
；The thickness of the section is 4 ~ 6 μm, and section, which is placed in 42 ~ 45 DEG C of water, opens up piece；The patch, roasting piece are：Slice is certainly
Taken out after so stretching, extension is flat from water, it is drained, dry 2 ~ 4h is placed at 37 ~ 45 DEG C, or spontaneously dries a night at room temperature；
Step（4）Described in dyeing dyed for H.E, operating procedure is：Section is sequentially placed into I 10min of dimethylbenzene, dimethylbenzene II
10min, 100% ethanol, I 5min, 100% ethanol, II 5min, 95% ethanol 5min, 85% ethanol 5min, 75% ethanol 5min, water
5min, hematoxylin 10 ~ 20min of dye liquor, flowing water rinse 5min, then are placed in HCl- ethanol differentiation 20 ~ 40s of liquid, and flowing water flushing 10 ~
30min, microscopy, flowing water rinses 2min, then is sequentially placed into 70% ethanol 1min, 85% ethanol 1min, eosin stain 2min, 95% second
I 2min of alcohol, 95% ethanol, II 2min, 100% ethanol, I 2min, 100% ethanol, II 2min, I 5min of dimethylbenzene, II 5min of dimethylbenzene.
2. preparation method according to claim 1, it is characterised in that：The hematoxylin dye liquor is：1g hematoxylins are dissolved in
Solution A is obtained in 100% ethanol of 20mL, 50g potassium alums, which are dissolved in 300mL distilled water, obtains second liquid；Solution A, second liquid are mixed, boil 2 ~
3min, then add distilled water to be settled to 1000mL；It is eventually adding 0.2g sodium iodates, to obtain the final product.
3. preparation method according to claim 1, it is characterised in that：The eosin stain is：0.5g Yihong is added to
In 85% ethanol of 100mL, dissolving is complete, to obtain the final product.
4. preparation method according to claim 1, it is characterised in that：The HCl- ethanol breaks up liquid：1mL hydrochloric acid is added
Enter into 70% ethanol of 99mL, mix, to obtain the final product.
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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 |
| CN114689407B (en) * | 2022-04-14 | 2023-05-19 | 四川大学华西医院 | Method for manufacturing animal micro tissue paraffin section |
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