CN105241686B - The preparation method of Hynobiidae animal retina microscopic tissue sections - Google Patents
The preparation method of Hynobiidae animal retina microscopic tissue sections Download PDFInfo
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- CN105241686B CN105241686B CN201510487763.8A CN201510487763A CN105241686B CN 105241686 B CN105241686 B CN 105241686B CN 201510487763 A CN201510487763 A CN 201510487763A CN 105241686 B CN105241686 B CN 105241686B
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Abstract
The invention discloses a kind of preparation method of Hynobiidae animal retina microscopic tissue sections, belong to microscopic tissue sections technical field.This method comprises the following steps：(1) eyeball of Hynobiidae animal is intactly taken out, and trims excess tissue；(2) eyeball of collection is placed in fixer, is rinsed after fixed spare；(3) eyeball tissue through fixing process is immersed in concentration gradient ethanol successively and be dehydrated, each 45~60min of serial dehydration；(4) eyeball 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；(5) eyeball 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；(6) eyeball 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 retina microscopic tissue sections, and in particular to for micro- sight
The preparation method for the Hynobiidae animal retina histotomy examined, belongs to microscopic tissue sections technical field.
Background technology
Important composition original paper of the eyes as vision, extraneous picture signal objectively can be converted into physiologic pulse and
Identified by central brain nerve, analyzed by nervous centralis and instruct animal to make various actions and behavior reaction with it.View
It is particularly important that film is located at chordate animal eyeball close to the back of brain side, and retina is in whole eyeball, because retina is held
Carry on a shoulder pole the whole task for converting optical signal into nerve signal.Different animals are due to activity pattern, habitat and evolve ground
, there is panoramic adaptive behavior in the difference of position.
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, active behavior can be divided into confused type or
Nocturnal habit, since the difference of living environment and active behavior causes the difference of illumination, so as to cause the change of retinal morphology structure
Change.The Morphologic Characteristics of retina are studied, the making of retina paraffin section is an essential step.
The content of the invention
The object of the present invention is to provide a kind of preparation method of Hynobiidae animal retina microscopic tissue sections.
In order to realize the above object the technical solution adopted in the present invention is：
The preparation method of Hynobiidae animal microscopic tissue sections, comprises the following steps：
(1) draw materials：The eyeball of Hynobiidae animal is intactly taken out, and trims excess tissue；
(2) fixed, flushing：The eyeball of collection is placed in fixer, is rinsed after fixed spare；
(3) it is dehydrated：By the eyeball 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；
(4) it is transparent：Eyeball tissue through dehydration is immersed to 2~3min in the mixed liquor of 100% ethanol and dimethylbenzene,
10~30min in dimethylbenzene is immersed after taking-up again；
(5) waxdip：Eyeball tissue 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；
(6) eyeball tissue handled through waxdip is embedded, sliced successively, exhibition piece, patch, roasting piece, dyeing, at sealing
Reason, to obtain the final product.
Fixer described in step (2) is 10% formalin solution, and dosage is 10~20 times of eyeball tissue volume.
It is dehydrated described in step (3) and carries out at room temperature.
The volume ratio of 100% ethanol and dimethylbenzene described in step (4) is 1:1.
The volume ratio of dimethylbenzene and paraffin described in step (5) is 1:1.
The thickness cut into slices described in step (6) is 4~6 μm, and section, which is placed in 42~45 DEG C of water, opens up piece.
Patch described in step (6), 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.
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.
Dyeing is dyed for H.E described in step (6), 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.
Beneficial effects of the present invention：
The preparation method of Hynobiidae animal retina microscopic tissue sections includes materials, fixed, flushing, takes off in the present invention
Water, transparent, waxdip, embedding, finishing, section and exhibition piece, patch and roasting piece, H.E dyeing, sealing, flow is simple, operation
Convenient, the section of acquisition is complete, and picture structure is clear, provides technical support for research Hynobiidae animal retina structure, fits
For research of the laboratory research worker to Hynobiidae animal retina histology and morphology structure.
Brief description of the drawings
Fig. 1 is Batrachuperus pinchonii (Batrachuperuspinchonii) retina 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.
Hynobiidae animal Batrachuperus pinchonii (Batrachuperuspinchonii) retina microscopic tissue sections in the present embodiment
Preparation method, comprise the following steps：
(1) draw materials：At room temperature, the eyeball of Hynobiidae animal is intactly taken out with scalpel and surgical clamp, and
Unnecessary tissue is trimmed；
(2) it is fixed：The eyeball of collection is put into 10% formalin fixer and is fixed, dosage is eyeball tissue body
Long-pending 15 times, carry out mark in order to identify；
(3) rinse：Eyeball tissue after fixation water is rinsed overnight；
(4) it is dehydrated：At room temperature, ethanol dehydration agent is made into different gradient concentrations with distilled water, then by eyeball 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, and the time 50min of ethanol dehydrations at different levels (need to such as be stayed overnight, eyeball should rest on 70% second
In alcohol)；
(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 eyeball is put into volume ratio 1:25min in 1 dimethylbenzene and the mixed liquor of paraffin, then
It is put at twice in 58 DEG C of pure wax again, each waxdip 50min, the common saturating waxes of 100min finish；
(7) embed：The paraffin of thawing is injected in embedded box, the eyeball of waxdip is inserted wherein, makes wax stone cold rapidly
Hard solid, embedded eyeball can preserve for a long time in wax stone；
(8) trimming of paraffin mass：Paraffin mass on embedded box is done into appropriate trimming；
(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：Retinal tissue edge drop neutral gum after dyeing, holds thin tweezer with the right hand and lightly clamps lid
The right side of slide, and its left side is placed on the gummy left side, then left hand holds the left side that dissecting needle buttresses coverslip, and the right hand will
Tweezers, which unclamp, to be gradually reduced, and is slowly extracted out, and so natural gum is unrolled evenly and will wherein bubble discharge in coverslip, uses dimethylbenzene
The natural gum that coverslip Zhou Duoyu sides are overflow is wiped clean, section is placed steadily, slightly weight, treats the dry solidification of natural gum, to obtain the final product, section
It can preserve for a long time.
Hynobiidae animal Batrachuperus pinchonii (Batrachuperuspinchonii) retina paraffin section is micro- in the present embodiment
For figure as shown in Figure 1, Lens is crystalline lens in figure, Retina is retina.
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.
Claims (4)
1. the preparation method of Hynobiidae animal retina microscopic tissue sections, it is characterised in that：Comprise the following steps：
（1）Materials：The eyeball of Hynobiidae animal is intactly taken out, and trims excess tissue；
（2）Fixed, flushing：The eyeball of collection is placed in fixer, is rinsed after fixed spare；
（3）Dehydration：Eyeball tissue through fixing process is immersed into concentration gradient 50%, 70%, 80%, 90%, 95%, 100% successively
It is dehydrated in ethanol, wherein 95%, 100% ethanol is respectively repeated twice, each 45 ~ 60min of serial dehydration；
（4）It is transparent：Eyeball tissue 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；
（5）Waxdip：Eyeball tissue 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；
（6）The eyeball tissue 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；
The dyeing is dyed for H.E, and operating procedure is：Section is sequentially placed into I 10min of dimethylbenzene, II 10min of dimethylbenzene, 100% second
I 5min of alcohol, 100% ethanol, II 5min, 95% ethanol 5min, 85% ethanol 5min, 75% ethanol 5min, water 5min, hematoxylin dye liquor
10 ~ 20min, flowing water rinse 5min, then are placed in HCl- ethanol differentiation 20 ~ 40s of liquid, and flowing water rinses 10 ~ 30min, microscopy, flowing water punching
2min is washed, 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.
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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| CN109884019B (en) * | 2019-03-25 | 2022-05-27 | 大连大学 | Three-dimensional curved surface reconstruction method applicable to biological membrane |
| CN110161436A (en) * | 2019-06-14 | 2019-08-23 | 山东师范大学 | A kind of Magnetic sensing methods of krill compound eye |
| CN110361242B (en) * | 2019-08-14 | 2022-01-18 | 武汉赛维尔生物科技有限公司 | Fixing liquid for eyeball tissue and pretreatment method for preparing eyeball tissue slices |
| CN110672395A (en) * | 2019-11-19 | 2020-01-10 | 李雄 | HE staining method for tissue section |
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