CN105571916A - Acipenser dabryanus oosperm tissue slicing method - Google Patents
Acipenser dabryanus oosperm tissue slicing method Download PDFInfo
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Abstract
The present invention relates to a microstructure slicing technology, and specifically relates to an acipenser dabryanus oosperm tissue slicing method. The acipenser dabryanus oosperm tissue slicing method mainly comprises seven steps of material fixation, dehydration, transparentizing, wax dipping, embedding, slicing and staining; a fixing liquid used in the material fixation is a Bouin's fixing liquid and a formaldehyde fixing liquid with the mass fraction of 10%; and the total dehydration time is 10-20h. The acipenser dabryanus oosperm tissue slicing method aims at sturgeon oosperm which belongs to a viscid egg, has the characteristics of being rich in vitellogenin, tough and dense in egg envelope, narrow in perivitelline space, and the like, a complete serial tissue slicing method suitable for sturgeon oosperm is established, the sturgeon oosperm can be efficiently processed, and better paraffin sections can be made.
Description
Technical field
The present invention relates to microscopic tissue sections technology, is exactly a kind of tissue section method of acipenser dabryanus embryonated egg specifically.
Background technology
Acipenser dabryanus (Acipenserdabryanus) is also known as acipenser dabryanus and Sha Lazi, that China distinctive settling down property of fresh water Acipenseridae (Acipenseridae) sturgeon belongs to fish, be distributed in Upper Yangtze River and downstream, Jinsha jiang River (fourth auspicious magnificent 1994), I grade, country lays special stress on protecting wild animal and Upper Yangtze River one-level protects fish (Liu Jun 2004) in a hurry, International Union for Conservation of Nature and Natural Resources (WorldConservationUnion, IUCN) pole danger level (criticallyendangered, CR) species (Du Jun etc. 2009), international endangered animal and plant kind trade pact (ConventiononInternationalTradeinEndangeredSpeciesofWildF aunaandFlora, CITES) appendix II protection species, one of main object of protection of Upper Yangtze River rare Endemic fish Nature Reserve.In recent years, due to the deterioration of Upper Yangtze River rivers ecologic environment, the exploitation of step hydropower station, natively less acipenser dabryanus resource declines further, existing is difficult to find, more difficultly catches.In order to protect its living resources better, recover Population, stablize ecologic environment, the reproductive study of sturgeons seems especially important.In addition when ensureing population quantity, acipenser dabryanus fish-egg can be used as the important source material of caviar, has higher economic worth, has certain contribution to economic development and raising people's living standard.Therefore, study acipenser dabryanus embryonated egg and there is very positive effect.
For the histotomy technology of fish oosperm, mainly using the embryonated egg of freshwater fish as experiment material, and the cyprinid fish mainly chosen, the embryonated egg microtomy of such as grass carp and crucian, also less seawater fish embryonated egg microtomy is had, such as left-eyed flounder.And report be there is not yet for the correlation technique of sturgeons embryonated egg.The method of freshwater fish is by its embryonated egg Bouin ' s immobile liquid or the fixing rear striping of Smith immobile liquid, according to general tissue section method through Gradient elution using ethanol, after the steps such as dimethylbenzene is transparent, paraffin embedding, carry out serial section, make paraffin section.And seawater fish is on the basis with reference to general dicing method, punctures egg membrane and agar embedding step through having gone before dewatering, enhancing dehydration and location.
On acipenser dabryanus, use above-mentioned two kinds of methods to the embryonated egg of its different development stage through row relax, the situation of appearance is often that histotomy is broken, and Yolk drops, and egg membrane fracture, separation, can not obtain than more complete section.Its main cause is that acipenser dabryanus ovum ovum footpath is larger, the tough and tensile densification of egg membrane, the all gaps of ovum after fixing its ovum compared with most of freshwater fish are very narrow and small, can not intactly striping, and owing to ovum week covering the thicker mucous membrane of one deck, dehydration, transparent, waxdip process in abundant not as good as other fish process, in the difference of the principal ingredient of embryonated egg different development stage, the successful difficulty that causes cutting into slices is very big.
Summary of the invention
The technical matters that the present invention solves is that acipenser dabryanus ovum ovum footpath is larger, the tough and tensile densification of egg membrane, the all gaps of ovum after fixing its ovum compared with most of freshwater fish are very narrow and small, can not intactly striping, and owing to belonging to viscid egg, its ovum week covers the thicker mucous membrane of one deck, dehydration, transparent, waxdip process in abundant not as good as other fish process, general fish oosperm tissue section method is not suitable for sturgeons, is sliced into power lower.
For solving the problem, we comprise the trial of method through the experiment of system, the optimization of condition, the adjustment of time, and a large amount of repeat experiment, establish the Serial tissue sections method being applicable to sturgeons embryonated egg of complete set, establish method and the condition of applicable sturgeons embryonated egg histotomy, be directed to sturgeon oosperm and belong to viscid egg, Yolk enriches, the tough and tensile densification of egg membrane, the features such as all gaps of ovum are narrow and small, effectively process embryonated egg and produce good paraffin section.
Technical scheme of the present invention is fixed primarily of material, dewater, transparent, waxdip, embedding, section and dyeing seven steps form; The immobile liquid that employing fixed by described material to be Bouin ' s immobile liquid or massfraction be 10% formaldehyde immobile liquid; Described dewatering time is 1-2h.Described transparent step after transparent liquid process, then processes 5-7h, to the transparent state of sturgeon ovum respectively through terpinol first time process 4-5h and terpinol second time; Described dehydration T.T. is 10-20h; The described waxdip time is 16-30h.
Described material fixing step be fresh sturgeon ovum is put into Bouin ' the s immobile liquid of 40-60 times of volume or volume fraction be 10% formaldehyde immobile liquid fix 20-30 hour after, be that 70% ethanol rinses repeatedly by volume fraction, then being kept at volume fraction is in the ethanol of 70%, changing a volume fraction every one day is the alcohol of 70%, saves backup after continuous three times.
Preferably, described material fixing step be fresh sturgeon ovum is put into 50 times of volumes the fixing or 10% formaldehyde immobile liquid of Bouin ' s immobile liquid fix 24 hours after, be kept at after repeatedly rinsing with 70% ethanol in 70% ethanol, 70% alcohol is changed every one day, continuous three times, fully to wash away immobile liquid, save backup.
Described dehydration is that the sturgeon ovum stored in 70% ethanol is directly immersed 80% ethanol dehydration once, time 2h, and passes through ethanol dehydration 2h of 90% successively, the ethanol dehydration twice of 95%, each 1-2h, the ethanol dehydration twice of 100%, each 1h.
Described transparent step is first the sturgeon ovum after dehydration is changed to the transparent liquid or terpinol that absolute ethyl alcohol mixes with terpinol equal-volume: dimethylbenzene: process 1-3h in the mixed transparent transition liquid of absolute ethyl alcohol=4:1:5, and then process 5-7h, to the transparent state of sturgeon ovum respectively through terpinol first time process 4-5h and terpinol second time.
Described waxdip step is first changed to by the sturgeon ovum after transparent processing in waxdip liquid that terpinol mixes with paraffin equal-volume to process 1-2h, and then processes 10-20h respectively through paraffin first time process 5-8h and paraffin second time.
Described embedding step is the sturgeon ovum conventional organization embedding method after being completed by waxdip, namely embeds with carton, makes paraffin mass.
Described section and staining procedure are by the continuous paraffin section of microtome, and through exhibition sheet, paster, roasting sheet, by haematoxylin & eosin (HE) dyeing, last microexamination is taken pictures.Concrete steps are for using microtome, and slice thickness controls at 5 μm-7 μm, extends piece in 35 DEG C of-38 DEG C of water-baths, after dragging for sheet, in the baking oven of 35 DEG C-38 DEG C, carries out baking sheet with the microslide scribbling albumen glycerine.
Haematoxylin & eosin (HE) decoration method concrete steps are as follows: first use first time dimethylbenzene process 10-15min, second time dimethylbenzene process 10-13min, be the dimethylbenzene of 1:1 by volume ratio again: absolute ethyl alcohol process 2min, the Ethanol Treatment 2min of first time 100%, the Ethanol Treatment 2min of second time 100%, the Ethanol Treatment 2min of first time 95%, the Ethanol Treatment 2min of second time 95%, the Ethanol Treatment 2min of 80%, the Ethanol Treatment 2min of 70%, brazilwood extract dyeing 20min, tap water 3min, 1% hydrochloride alcohol solution process 5s, WITH AMMONIA TREATMENT 15s after washing, wash again, the Ethanol Treatment 3min of 70%, the Ethanol Treatment 3min of 80%, the Ethanol Treatment 3min of 90%, eosin stains 30s, the Ethanol Treatment 4min of 95%, the Ethanol Treatment 4min of first time 100%, the Ethanol Treatment 4min of second time 100%, be the absolute ethyl alcohol of 1:1 by volume ratio: dimethylbenzene process 5min, first time dimethylbenzene process 5min, second time dimethylbenzene process 5min makes it transparent, then uses neutral gum mounting.
As can be seen from accompanying drawing 1-4, adopt acipenser dabryanus fish fertilized egg histotomy prepared by the inventive method, egg membrane is complete, and histotomy is in flakes complete, and without incomplete, overlapping, institutional framework is clear, well arranged, and cell boundary line is clear, and staining versus is obvious, has no ash dye.
The invention has the beneficial effects as follows:
1. the present invention establishes method and the condition of applicable sturgeons embryonated egg histotomy, belong to viscid egg for sturgeon oosperm, Yolk enriches, the tough and tensile densification of egg membrane, the features such as all gaps of ovum are narrow and small, the present invention effectively can process embryonated egg and produce good paraffin section.The embryonated egg having scholar to think that Bouin ' s immobile liquid is fixed there will be the hard and crisp situation of Yolk, to such an extent as to can not serial section, also the effect that Smith immobile liquid is fixed is better than Bouin ' s immobile liquid to have scholar to think, be not easy to make embryonated egg hardening crisp, but in the present invention, use Bouin ' s immobile liquid or 10% formaldehyde immobile liquid process about 24h that Yolk can not be made hardening crisp, can serial section.Therefore, the present invention can reduce the troublesome operation step using Smith immobile liquid to bring.
2. the present invention is by a large amount of explorations to dewatering time, determines best dewatering time, fully can not take off water problem when solving most of embryonated egg histotomy.Because sturgeons embryonated egg belongs to viscid egg, the mucous membrane that its periphery has one deck thicker, in addition its one-level egg membrane and all tough and tensile densification of secondary egg membrane, if dewatering time is not reserved, the easy sharply catastrophic collapse when dewatering being caused, causing ovum week uneven, impact section.Conventional method is that the method peeling egg membrane off solves this problem, but all gaps of sturgeons embryonated egg ovum are very narrow and small, can not divest, also the method by punching on egg membrane with dissecting needle is had to solve, but all add operation steps, and the present invention is by the control of dewatering time, need not carry out special process to egg membrane, not only reach the object of dewatering completely, and the integrality of the egg membrane retained.
3. this experiment is in multiple periods of sturgeons embryonated egg all through having gone identical disposal route, and be obtained for complete histotomy, repetition rate is high, effective, and therefore the present invention is applicable to the histotomy in each period of sturgeons embryonated egg.
4. the present invention is simple to operate, rationally proper, is not only applicable to the histotomy of sturgeons embryonated egg, is also applicable to viscid egg, the embryonated egg histotomy of other fish megalecithal, applied widely.
Accompanying drawing explanation
Fig. 1: the single complete ovum section that the cleavage stage histotomy that the application embodiment of the present invention 1 carries out acipenser dabryanus embryonated egg obtains;
Fig. 2: the egg membrane that the cleavage stage histotomy that the application embodiment of the present invention 1 carries out acipenser dabryanus embryonated egg obtains amplifies photograph;
Fig. 3: the application embodiment of the present invention 2 carries out the histotomy slice map in primitive gut period of acipenser dabryanus embryonated egg;
Fig. 4: the application embodiment of the present invention 3 carries out acipenser dabryanus embryonated egg phase histotomy figure;
Fig. 5: Comparison study embodiment 1 carries out acipenser dabryanus embryonated egg histotomy gastrul stage figure;
Fig. 6: Comparison study embodiment 2 carries out acipenser dabryanus embryonated egg embryonated egg phase histotomy egg membrane figure;
Fig. 7: Comparison study embodiment 2 carries out acipenser dabryanus embryonated egg embryonated egg phase histotomy figure.
Embodiment
For making the object of the embodiment of the present invention, technical scheme and advantage clearly, below in conjunction with the accompanying drawing in the embodiment of the present invention, technical scheme in the embodiment of the present invention is clearly and completely described, obviously, described embodiment is the present invention's part embodiment, instead of whole embodiments.Based on the embodiment in the present invention, those of ordinary skill in the art, not making the every other embodiment obtained under creative work prerequisite, belong to the scope of protection of the invention.
Embodiment 1:
A tissue section method for acipenser dabryanus embryonated egg cleavage stage, is made up of following steps:
Material is fixed: fix 24h by growing Bouin ' the s immobile liquid putting into 50 times of volumes to the acipenser dabryanus ovum of embryonated egg cleavage stage, repeatedly rinse with the ethanol that volume fraction is 70%, then be kept in 70% ethanol, 70% alcohol is changed every one day, continuous three times, fully to wash away immobile liquid, save backup.
Dehydration: the embryonated egg be kept in 70% ethanol is carried out serial dehydration, 80% ethanol dehydration one time 2h, 90% ethanol dehydration 2h, 95% ethanol dehydration twice, each 2h, then use 100% ethanol dehydration twice, respectively dewater 1h at every turn.
Transparent: the complete embryonated egg of dehydration to be inserted volume fraction terpinol: process 1h in the transparent transition liquid of absolute ethyl alcohol=1:1, then process 5h with first time in terpinol respectively, second time processes 8h.
Waxdip: whole process is carried out in 57 DEG C of constant temperature ovens.First insert in the waxdip liquid of terpinol and 57 DEG C of paraffin equal-volume mixing and process 1h, and then process 4h respectively through paraffin first time, paraffin second time processes 10h.
Embedding: the sturgeon ovum conventional organization embedding method after being completed by waxdip, namely embeds with carton, make paraffin mass.
Section: histotomy, carries out conventional process after section, by the continuous paraffin section of microtome, for subsequent use through exhibition sheet, paster, roasting sheet aftertreatment.
Dyeing: haematoxylin & eosin (HE) dyes, mounting 37 DEG C of constant temperature ovens are dried, and finally observe and take pictures (as Fig. 1-2).
Embodiment 2:
Tissue section method gastrul stage for acipenser dabryanus embryonated egg, is made up of following steps:
Material is fixed: by grow to the acipenser dabryanus embryonated egg of different times put into massfraction be 10% formaldehyde immobile liquid fix 24h, repeatedly rinse with the ethanol that volume fraction is 70%, then be kept in 70% ethanol, 70% alcohol is changed every one day, continuous three times, fully to wash away immobile liquid, save backup.
Dehydration: the embryonated egg be kept in 70% ethanol is carried out serial dehydration, 80% ethanol dehydration one time 2h, 90% ethanol dehydration 2h, 95% ethanol dehydration twice, each 2h, then use 100% ethanol dehydration twice, respectively dewater 1h at every turn.
Transparent: the complete embryonated egg of dehydration to be inserted volume fraction terpinol: process 2h in the transparent transition liquid of absolute ethyl alcohol=1:1, then process 4h with first time in terpinol respectively, second time processes 6h.
Waxdip: whole process is carried out in 57 DEG C of constant temperature ovens.First insert in the waxdip liquid of terpinol and 57 DEG C of paraffin equal-volume mixing and process 1h, and then process 5h respectively through paraffin refined wax first time, paraffin refined wax second time processes 20h.
Embedding: the sturgeon ovum conventional organization embedding method after being completed by waxdip, namely embeds with carton, make paraffin mass.
Section: histotomy, carries out conventional process after section, by the continuous paraffin section of microtome, for subsequent use through exhibition sheet, paster, roasting sheet aftertreatment.
Dyeing: haematoxylin & eosin (HE) dyes, mounting 37 DEG C of constant temperature ovens are dried, and finally observe and take pictures (as Fig. 3).
Embodiment 3:
The tissue section method of acipenser dabryanus embryonated egg embryonated egg phase, is made up of following steps:
Material is fixed: fix 24h by growing Bouin ' the s immobile liquid putting into 50 times of volumes to the acipenser dabryanus embryonated egg of different times, repeatedly rinse with the ethanol that volume fraction is 70%, then be kept in 70% ethanol, 70% alcohol is changed every one day, continuous three times, fully to wash away immobile liquid, save backup.
Dehydration: the embryonated egg be kept in 70% ethanol is carried out serial dehydration, 80% ethanol dehydration one time 2h, 90% ethanol dehydration 2h, 95% ethanol dehydration twice, each 1h, then use 100% ethanol dehydration twice, respectively dewater 1h at every turn.
Transparent: the complete embryonated egg of dehydration to be inserted volume fraction terpinol: process 2h in the transparent transition liquid of absolute ethyl alcohol=1:1, then process 5h with first time in terpinol respectively, second time processes 5h.
Waxdip: whole process is carried out in 57 DEG C of constant temperature ovens.First insert in the waxdip liquid of terpinol and 57 DEG C of paraffin equal-volume mixing and process 1h, and then process 5h respectively through paraffin first time, paraffin second time processes 10h.
Embedding: the sturgeon ovum conventional organization embedding method after being completed by waxdip, namely embeds with carton, make paraffin mass.
Section: histotomy, carries out conventional process after section, by the continuous paraffin section of microtome, for subsequent use through exhibition sheet, paster, roasting sheet aftertreatment.
Dyeing: haematoxylin & eosin (HE) dyes, mounting 37 DEG C of constant temperature ovens are dried, and finally observe and take pictures (as Fig. 4).
Comparative example 1:
The tissue section method of sturgeons embryonated egg gastrul stage, is made up of following steps:
Step one: fix 24h by growing Bouin ' the s immobile liquid putting into 50 times of volumes to the acipenser dabryanus embryonated egg of different times, repeatedly rinse with the ethanol that volume fraction is 70%, then be kept in 70% ethanol, 70% alcohol is changed every one day, continuous three times, fully to wash away immobile liquid, save backup.
Step 2: the embryonated egg be kept in 70% ethanol is carried out serial dehydration, 80% ethanol dehydration one time 2h, 90% ethanol dehydration 2h, 95% ethanol dehydration twice, each 2h, then use 100% ethanol dehydration twice, respectively dewater 1h at every turn.
Step 3: the complete embryonated egg of dehydration is inserted volume fraction terpinol: process 0.5h in the transparent transition liquid of absolute ethyl alcohol=1:1, then process 15min with first time in terpinol respectively, second time processes 15min.
Step 4: whole process is carried out in 57 DEG C of constant temperature ovens.First insert in the waxdip liquid of terpinol and 57 DEG C of paraffin equal-volume mixing and process 1h, and then process 2h respectively through paraffin first time, paraffin second time processes 2h.
Step 5: carry out paraffin embedding, make paraffin mass.
Step 6: histotomy, carries out conventional process after section, haematoxylin & eosin (HE) dyes, and mounting 37 DEG C of constant temperature ovens are dried, and finally observes and takes pictures (as Fig. 5).
Comparative example 2:
The tissue section method of sturgeons embryonated egg embryonated egg phase, is made up of following steps:
Step one: fix 24h by growing Bouin ' the s immobile liquid putting into 50 times of volumes to the acipenser dabryanus embryonated egg of different times, repeatedly rinse with the ethanol that volume fraction is 70%, then be kept in 70% ethanol, 70% alcohol is changed every one day, continuous three times, fully to wash away immobile liquid, save backup.
Step 2: poke egg membrane on embryonated egg surface with dissecting needle, is placed in double dish, and the warm agar of preparation 1%, slowly pours double dish into, cover embryonated egg, naturally cools, is cut into into agar block with scalpel.
Step 2: the agar block containing embryonated egg is carried out serial dehydration, 80% ethanol dehydration one time 2h, 90% ethanol dehydration 2h, 95% ethanol dehydration twice, each 2h, then use 100% ethanol dehydration twice, respectively dewater 1h at every turn.
Step 3: the complete agar block of dehydration is inserted dimethylbenzene: 15min in the transparent transition liquid of absolute ethyl alcohol=1:1, then process 10min with first time in terpinol respectively, second time processes 5min.
Step 4: waxdip, whole process is carried out in 57 DEG C of constant temperature ovens.First insert 1h in the mixed liquid such as terpinol and 57 DEG C of paraffin, and then process 1.5h respectively through paraffin first time, paraffin second time processes 1.5h.
Step 5: carry out paraffin embedding, make paraffin mass.
Step 6: histotomy, carries out conventional process after section, haematoxylin & eosin (HE) dyes, and mounting 37 DEG C of constant temperature ovens are dried, and finally observes and takes pictures (as Fig. 6 and 7).
As can be seen from accompanying drawing 6-7, the section egg membrane fracture adopting comparative example to obtain, Yolk disperse, egg membrane is separated with Yolk, and egg membrane ruptures, and can not reach requirement.
Claims (8)
1. a tissue section method for acipenser dabryanus embryonated egg, is characterized in that: fixed by material, dewater, transparent, waxdip, embedding, section and dyeing seven steps form; Described transparent step after transparent liquid process, then processes 5-7h, to the transparent state of sturgeon ovum respectively through terpinol first time process 4-5h and terpinol second time; Described dehydration T.T. is 10-20h; The described waxdip time is 16-30h.
2. the tissue section method of a kind of acipenser dabryanus embryonated egg as claimed in claim 1, it is characterized in that: described material fixing step be fresh acipenser dabryanus fish-egg is put into Bouin ' the s immobile liquid of 40-60 times of volume or massfraction be 10% formaldehyde immobile liquid fix 20-30 hour after, repeatedly rinse with 70% ethanol, then being kept at volume fraction is in the ethanol of 70%, changing a volume fraction every one day is 70% ethanol, saves backup after continuous three times in 70% ethanol.
3. the tissue section method of a kind of acipenser dabryanus embryonated egg as claimed in claim 1 or 2, it is characterized in that: described material fixing step be fresh acipenser dabryanus fish-egg is put into 50 times of volumes the fixing or 10% formaldehyde immobile liquid of Bouin ' s immobile liquid fix 24 hours after, be kept at after repeatedly rinsing with 70% ethanol in 70% ethanol, changed 70% alcohol every one day, save backup after continuous three times.
4. the tissue section method of a kind of acipenser dabryanus embryonated egg as claimed in claim 1, it is characterized in that: described dehydration is that the sturgeon ovum stored in 70% ethanol is directly immersed 80% ethanol dehydration once, time 2h, and pass through ethanol dehydration 2h of 90% successively, the ethanol dehydration twice of 95%, each 1-2h, the ethanol dehydration twice of 100%, each 1-2h.
5. the tissue section method of a kind of acipenser dabryanus embryonated egg as claimed in claim 1, it is characterized in that: described transparent step is that first by the sturgeon ovum after dehydration, to insert volume ratio be terpinol: the transparent transition liquid of absolute ethyl alcohol=1:1 or volume ratio are terpinol: dimethylbenzene: process 1-2h in the mixed transparent transition liquid of absolute ethyl alcohol=4:1:5, and then process 5-7h, to the transparent state of sturgeon ovum respectively through terpinol first time process 4-5h and terpinol second time.
6. the tissue section method of a kind of acipenser dabryanus embryonated egg as claimed in claim 1, it is characterized in that: described waxdip step is first changed to by the sturgeon ovum after transparent processing in waxdip liquid that terpinol mixes with paraffin equal-volume to process 1-2h, and then processes 10-20h respectively through paraffin first time process 5-8h and paraffin second time; Described waxdip process is carried out in 57 DEG C of constant temperature ovens.
7. the tissue section method of a kind of acipenser dabryanus embryonated egg as claimed in claim 1, is characterized in that: described embedding step is the sturgeon ovum conventional organization embedding method after being completed by waxdip, namely embeds with carton, makes paraffin mass.
8. the tissue section method of a kind of acipenser dabryanus embryonated egg as claimed in claim 1, it is characterized in that: described section and staining procedure are by the continuous paraffin section of microtome, through exhibition sheet, paster, roasting sheet, by haematoxylin & eosin (HE) dyeing, last microexamination is taken pictures.
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| CN110361242A (en) * | 2019-08-14 | 2019-10-22 | 武汉赛维尔生物科技有限公司 | It is a kind of for the fixer of eyeball tissue and the preprocess method of eyeball tissue film-making |
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| CN110361242A (en) * | 2019-08-14 | 2019-10-22 | 武汉赛维尔生物科技有限公司 | It is a kind of for the fixer of eyeball tissue and the preprocess method of eyeball tissue film-making |
| 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 |
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