CN110823663A - Method for manufacturing pathological section of animal eyeball - Google Patents
Method for manufacturing pathological section of animal eyeball Download PDFInfo
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- CN110823663A CN110823663A CN201910954209.4A CN201910954209A CN110823663A CN 110823663 A CN110823663 A CN 110823663A CN 201910954209 A CN201910954209 A CN 201910954209A CN 110823663 A CN110823663 A CN 110823663A
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- 210000005252 bulbus oculi Anatomy 0.000 title claims abstract description 98
- 241001465754 Metazoa Species 0.000 title claims abstract description 30
- 230000001575 pathological effect Effects 0.000 title claims abstract description 23
- 238000000034 method Methods 0.000 title claims abstract description 17
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 11
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 73
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims abstract description 30
- 239000007788 liquid Substances 0.000 claims abstract description 13
- 229930040373 Paraformaldehyde Natural products 0.000 claims abstract description 10
- 229920002866 paraformaldehyde Polymers 0.000 claims abstract description 10
- 238000007598 dipping method Methods 0.000 claims abstract description 6
- 239000011521 glass Substances 0.000 claims abstract description 6
- 239000012188 paraffin wax Substances 0.000 claims description 24
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 claims description 18
- 239000003153 chemical reaction reagent Substances 0.000 claims description 18
- 239000008096 xylene Substances 0.000 claims description 18
- 238000005520 cutting process Methods 0.000 claims description 5
- 230000018044 dehydration Effects 0.000 claims description 5
- 238000006297 dehydration reaction Methods 0.000 claims description 5
- 210000003205 muscle Anatomy 0.000 claims description 5
- 239000001993 wax Substances 0.000 claims description 5
- 210000004087 cornea Anatomy 0.000 claims description 4
- 210000001525 retina Anatomy 0.000 abstract description 6
- 230000009286 beneficial effect Effects 0.000 abstract description 3
- 210000001519 tissue Anatomy 0.000 description 8
- 238000000926 separation method Methods 0.000 description 5
- 239000000834 fixative Substances 0.000 description 3
- 230000002207 retinal effect Effects 0.000 description 3
- 210000003754 fetus Anatomy 0.000 description 2
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 241000282693 Cercopithecidae Species 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 210000002718 aborted fetus Anatomy 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 210000000981 epithelium Anatomy 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/36—Embedding or analogous mounting of samples
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/42—Low-temperature sample treatment, e.g. cryofixation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
- G01N2001/2873—Cutting or cleaving
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
- G01N2001/305—Fixative compositions
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Abstract
The invention relates to the field of histopathology, and particularly discloses a method for manufacturing pathological sections of an animal eyeball, which comprises the following steps: (1) picking up eyeballs; (2) fixing eyeballs: placing the separated eyeballs into a fixing solution precooled to 4.0 ℃, and placing the fixing solution into a refrigerator set to 4.0 ℃ for fixing for 48 hours; (3) the eyeball sight glass is dehydrated and transparent; (4) wax dipping and embedding; (5) slicing; in the step (2), the fixing liquid comprises the following components in percentage by volume: 4% paraformaldehyde solution: anhydrous acetic acid: anhydrous ethanol ═ 10: (1.5-2.0): (8.0-8.5). The invention is beneficial to preparing high-quality pathological sections of the eyeball so as to promote the basic application of retina-related research.
Description
Technical Field
The invention relates to the field of histopathology, in particular to a method for manufacturing pathological sections of animal eyeballs.
Background
The pathological technology conventionally applied in the field of ophthalmologic research relates to the preparation of eyeball tissue specimens, and the eyeball specimens fixed by conventional fixative and conventional treatment flows are easy to have the phenomena of eyeball deformation and separation of tissues of each layer of the eyeball, particularly the separation of retinal tissues; the low-quality pathological section restricts the high-quality pathological research in the field of ophthalmic research; therefore, it is necessary to develop a new fixative specifically for animal eyeball and optimize the application procedure to prepare high quality pathological section of eyeball, which helps promote the basic application of retina-related research.
Disclosure of Invention
In view of this, the present invention aims to provide a method for manufacturing pathological sections of an animal eyeball, which is beneficial to preparing high-quality pathological sections of the eyeball so as to promote the basic application of retina-related research.
The invention provides a method for manufacturing pathological sections of animal eyeballs, which comprises the following steps:
(1) taking eyeballs: cutting off the muscles attached to the outside of the eyeball of the animal, and separating the eyeball;
(2) fixing eyeballs: placing the separated eyeballs into a fixing solution precooled to 4.0 ℃, and placing the fixing solution into a refrigerator set to 4.0 ℃ for fixing for 48 hours;
(3) the dehydration of the eyeball sight glass is transparent: treating the eyeball treated in the step (2) with the following reagents in sequence: 1h of 75% ethanol, 1h of 90% ethanol, 1h of 95% ethanol, 1-1.5h of absolute ethanol, 1-2h of xylene and 1-2h of xylene;
(4) wax dipping and embedding; treating the eyeball treated in the step (3) with the following reagents in sequence: paraffin for 1-2h and paraffin for 1-2 h; after treatment, paraffin embedding is carried out;
(5) slicing: slicing according to a set thickness;
in the step (2), the fixing liquid comprises the following components in percentage by volume: 4% paraformaldehyde solution: anhydrous acetic acid: anhydrous ethanol ═ 10: (1.5-2.0): (8.0-8.5).
As a further improvement of the technical solution of the present invention, in the step (2), the components and volume ratios of the fixing solution are as follows: 4% paraformaldehyde solution: anhydrous acetic acid: anhydrous ethanol ═ 10: 1.5: 8.5.
as a further improvement of the technical solution of the present invention, in step (3), the eyeballs treated in step (2) are sequentially treated with the following reagents: 1h of 75% ethanol, 1h of 90% ethanol, 1h of 95% ethanol, 1h of absolute ethanol, 1h of xylene and 1h of xylene;
in step (4), the eyeballs treated in step (3) are sequentially treated with the following reagents: paraffin wax 1h and paraffin wax 1 h.
As a further improvement of the technical solution of the present invention, in the step (2), the components and volume ratios of the fixing solution are as follows: 4% paraformaldehyde solution: anhydrous acetic acid: anhydrous ethanol ═ 10: 1.0: 8.0.
as a further improvement of the technical scheme of the invention, in the step (2), before the separated eyeball is put into the stationary liquid, a needle head is used for forming a plurality of small holes in the central part of the cornea of the eyeball.
As a further improvement of the technical solution of the present invention, in step (3), the eyeballs treated in step (2) are sequentially treated with the following reagents: 1h of 75% ethanol, 1h of 90% ethanol, 1h of 95% ethanol, 1.5h of absolute ethanol, 1.5h of xylene and 1.5h of xylene;
in step (4), the eyeballs treated in step (3) are sequentially treated with the following reagents: paraffin 2h and paraffin 2 h.
Compared with the prior art, the invention has the following beneficial technical effects:
according to the method for manufacturing the pathological section of the animal eyeball, the improved stationary liquid is used, the use program is optimized, the prepared pathological section of the eyeball is observed and displayed under a microscope after HE dyeing, all layers of tissues of the eyeball are closely connected, the structure is flat, the cell form is very good, and the arrangement is regular; therefore, the fixative and the corresponding program can maintain the very good structure of the eyeball tissues of the small animals and the large animals, avoid the separation and deformation of each layer of tissue, avoid the fixed retinal separation caused by the fixation of the conventional specified liquid at present, are very helpful to improve the quality of the preparation of the eyeball specimen, avoid the separation of the retina and the pigment epithelium, prepare high-quality paraffin and frozen sections, and better serve the research field of ophthalmology.
Drawings
FIG. 1 is a photograph of a mouse retina slice made according to one embodiment of the present invention under a quadruple objective lens;
FIG. 2 is a photograph of a mouse retina slice made according to one embodiment of the present invention under a forty-fold objective lens;
fig. 3 is a photograph of a retinal slice of an aborted fetus made in accordance with a second embodiment of the present invention under a quadruple objective.
Detailed Description
The invention will be described in detail below with reference to examples and figures, which are given by way of illustration only and are not limiting to the scope of application of the invention. The present invention is not limited to the following embodiments or examples, and modifications and variations made without departing from the spirit of the invention are intended to be included within the scope of the invention.
The invention provides a method for manufacturing pathological sections of animal eyeballs, which comprises the following steps:
(1) taking eyeballs: cutting off the muscles attached to the outside of the eyeball of the animal, and separating the eyeball;
(2) fixing eyeballs: placing the separated eyeballs into a fixing solution precooled to 4.0 ℃, and placing the fixing solution into a refrigerator set to 4.0 ℃ for fixing for 48 hours;
(3) the dehydration of the eyeball sight glass is transparent: treating the eyeball treated in the step (2) with the following reagents in sequence: 1h of 75% ethanol, 1h of 90% ethanol, 1h of 95% ethanol, 1.5h of absolute ethanol, 1.5h of xylene and 1.5h of xylene;
(4) wax dipping and embedding; treating the eyeball treated in the step (3) with the following reagents in sequence: paraffin for 2h and paraffin for 2 h; after treatment, paraffin embedding is carried out;
(5) slicing: slicing according to a set thickness;
in the step (2), the fixing liquid comprises the following components in percentage by volume: 4% paraformaldehyde solution: anhydrous acetic acid: anhydrous ethanol ═ 10: (1.5-2.0): (8.0-8.5).
Example one
The embodiment provides a method for manufacturing pathological sections of animal eyeballs, which comprises the following steps:
(1) taking eyeballs: cutting off the muscles attached to the outside of the eyeball of the small animal, and separating the eyeball; in this embodiment, the eyeball of a small animal is exemplified, and the small animal is a C57 mouse aged 8 weeks, but it is needless to say that other small animal eyeballs having a small eyeball wall thickness, for example, a rat eyeball, may be used.
(2) Fixing eyeballs: placing the separated eyeballs into a fixing solution precooled to 4.0 ℃, and placing the fixing solution into a refrigerator set to 4.0 ℃ for fixing for 48 hours; the fixing liquid comprises the following components in percentage by volume: 4% paraformaldehyde solution: anhydrous acetic acid: anhydrous ethanol ═ 10: (1.5-2.0): (8.0-8.5).
(3) The dehydration of the eyeball sight glass is transparent: treating the eyeball treated in the step (2) with the following reagents in sequence: 1h of 75% ethanol, 1h of 90% ethanol, 1h of 95% ethanol, 1h of absolute ethanol, 1h of xylene and 1h of xylene.
(4) Wax dipping and embedding; treating the eyeball treated in the step (3) with the following reagents in sequence: paraffin wax for 1h and paraffin wax for 1 h; after treatment, paraffin embedding was performed.
(5) Slicing: slicing according to a set thickness; set to a thickness of 4 microns and HE stained and visualized photographically.
As shown in figures 1 and 2, observation under a microscope after HE staining shows that tissues of each layer of the eyeball of the mouse are closely connected, the structure is smooth, the cell morphology is very good, and the arrangement is regular.
Example two
The embodiment provides a method for manufacturing pathological sections of animal eyeballs, which comprises the following steps:
(1) taking eyeballs: cutting off the muscles attached to the outside of the eyeball of the large animal, and separating the eyeball; in this embodiment, the eyeball of a large animal is taken as an example, the large animal is a fetus born in 24 weeks, and other large animal eyeballs with a large eyeball wall thickness, such as a rabbit eyeball, a cat eyeball, a pig eyeball, a monkey eyeball, etc., may be adopted.
(2) Fixing eyeballs: firstly, a needle head is used for forming a plurality of small holes in the central part of the cornea of an eyeball, for example, a 33G needle head is used for pricking 4 small holes in the central part of the cornea so as to accelerate the penetration of stationary liquid; then, placing the separated eyeballs into a fixing solution precooled to 4.0 ℃, and placing the fixing solution into a refrigerator with the temperature set to 4.0 ℃ for fixing for 48 hours; the fixing liquid comprises the following components in percentage by volume: 4% paraformaldehyde solution: anhydrous acetic acid: anhydrous ethanol ═ 10: 2.0: 8.0.
(3) the dehydration of the eyeball sight glass is transparent: treating the eyeball treated in the step (2) with the following reagents in sequence: 1h of 75% ethanol, 1h of 90% ethanol, 1h of 95% ethanol, 1h of absolute ethanol, 1h of xylene and 1h of xylene.
(4) Wax dipping and embedding; treating the eyeball treated in the step (3) with the following reagents in sequence: paraffin for 2h and paraffin for 2 h; after treatment, paraffin embedding was performed.
(5) Slicing: slicing according to a set thickness; set to a thickness of 4 microns and HE stained and visualized photographically.
As shown in figure 3, observation under a microscope after HE staining shows that tissues of each layer of 24-week-old fetus eyeball are tightly connected, the structure is smooth, the cell morphology is very good, and the arrangement is regular.
Finally, the principle and embodiments of the present invention are explained by using specific examples, and the above descriptions of the examples are only used to help understand the core idea of the present invention, and the present invention can be modified and modified without departing from the principle of the present invention, and the modified and modified examples also fall into the protection scope of the present invention.
Claims (6)
1. The method for manufacturing the pathological section of the animal eyeball is characterized by comprising the following steps:
(1) taking eyeballs: cutting off the muscles attached to the outside of the eyeball of the animal, and separating the eyeball;
(2) fixing eyeballs: placing the separated eyeballs into a fixing solution precooled to 4.0 ℃, and placing the fixing solution into a refrigerator set to 4.0 ℃ for fixing for 48 hours;
(3) the dehydration of the eyeball sight glass is transparent: treating the eyeball treated in the step (2) with the following reagents in sequence: 1h of 75% ethanol, 1h of 90% ethanol, 1h of 95% ethanol, 1-1.5h of absolute ethanol, 1-2h of xylene and 1-2h of xylene;
(4) wax dipping and embedding; treating the eyeball treated in the step (3) with the following reagents in sequence: paraffin for 1-2h and paraffin for 1-2 h; after treatment, paraffin embedding is carried out;
(5) slicing: slicing according to a set thickness;
in the step (2), the fixing liquid comprises the following components in percentage by volume: 4% paraformaldehyde solution: anhydrous acetic acid: anhydrous ethanol ═ 10: (1.5-2.0): (8.0-8.5).
2. The method for preparing pathological section of animal eyeball according to claim 1,
in the step (2), the components and volume ratio of the fixing liquid are as follows: 4% paraformaldehyde solution: anhydrous acetic acid: anhydrous ethanol ═ 10: 1.5: 8.5.
3. the method for preparing pathological section of animal eyeball according to claim 2,
in step (3), the eyeballs treated in step (2) are sequentially treated with the following reagents: 1h of 75% ethanol, 1h of 90% ethanol, 1h of 95% ethanol, 1h of absolute ethanol, 1h of xylene and 1h of xylene;
in step (4), the eyeballs treated in step (3) are sequentially treated with the following reagents: paraffin wax 1h and paraffin wax 1 h.
4. The method for preparing pathological section of animal eyeball according to claim 1,
in the step (2), the components and volume ratio of the fixing liquid are as follows: 4% paraformaldehyde solution: anhydrous acetic acid: anhydrous ethanol ═ 10: 1.0: 8.0.
5. the method for preparing pathological section of animal eyeball according to claim 4,
in the step (2), before the separated eyeball is put into the stationary liquid, a plurality of small holes are formed in the central part of the cornea of the eyeball by using the needle head.
6. The method for preparing pathological section of animal eyeball according to claim 4,
in step (3), the eyeballs treated in step (2) are sequentially treated with the following reagents: 1h of 75% ethanol, 1h of 90% ethanol, 1h of 95% ethanol, 1.5h of absolute ethanol, 1.5h of xylene and 1.5h of xylene;
in step (4), the eyeballs treated in step (3) are sequentially treated with the following reagents: paraffin 2h and paraffin 2 h.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN111238901A (en) * | 2020-03-04 | 2020-06-05 | 四川省人民医院 | Manufacturing method of improved mouse eyeball frozen section |
| CN112781960A (en) * | 2021-02-05 | 2021-05-11 | 四川省人民医院 | Method for making eye frozen section |
| CN114088493A (en) * | 2021-11-19 | 2022-02-25 | 四川大学华西医院 | Animal eyeball pathological section manufacturing method |
-
2019
- 2019-10-09 CN CN201910954209.4A patent/CN110823663A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN111238901A (en) * | 2020-03-04 | 2020-06-05 | 四川省人民医院 | Manufacturing method of improved mouse eyeball frozen section |
| CN111238901B (en) * | 2020-03-04 | 2023-03-03 | 四川省人民医院 | Manufacturing method of improved mouse eyeball frozen section |
| CN112781960A (en) * | 2021-02-05 | 2021-05-11 | 四川省人民医院 | Method for making eye frozen section |
| CN112781960B (en) * | 2021-02-05 | 2024-02-23 | 四川省人民医院 | Method for making frozen eye slices |
| CN114088493A (en) * | 2021-11-19 | 2022-02-25 | 四川大学华西医院 | Animal eyeball pathological section manufacturing method |
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Application publication date: 20200221 |