CN104483178B - The preparation method of epinephelus akaara adult fish chromosome - Google Patents
The preparation method of epinephelus akaara adult fish chromosome Download PDFInfo
- Publication number
- CN104483178B CN104483178B CN201410682981.2A CN201410682981A CN104483178B CN 104483178 B CN104483178 B CN 104483178B CN 201410682981 A CN201410682981 A CN 201410682981A CN 104483178 B CN104483178 B CN 104483178B
- Authority
- CN
- China
- Prior art keywords
- fish
- hypotonic
- early stage
- weight
- cell suspension
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Abstract
A kind of preparation method of epinephelus akaara adult fish chromosome, belongs to cell biology, and it includes material processing early stage, prepares renal cell suspension, Hypotonic treatment, fixation, drop piece and dyeing；Described material processing early stage is using phytohaemagglutinin internal injection method, by the dosage of 15 μ g/g fish bodies weight, PHA is injected to the pectoral fin base portion of experiment fish, weighs the dosage injection colchicine solution of 2 μ g/g fish bodies weight after 24h by fish body, after 2.5 3h, head-kidney is taken out.The present invention uses the ground method of shot phytohaemagglutinin, while increases injection concentration, just can obtain good effect after 24h, save the time.The present invention is hardly resulted in complete cell, therefore the change prior art of the invention when carrying out Hypotonic treatment by prior art, is obtained clearly chromosome picture due to the histocyte characteristic of epinephelus akaara.
Description
Technical field
The invention belongs to cell biology, more particularly to a kind of preparation method of red lithosporic adult fish chromosome.
Background technology
The characteristics of preparation manipulation of chromosome specimen is simple, cheap, experimental site requirement is low, is always traditional life
Thing genetics research technology, prepared by chromosome specimen include：Pre-treatment, Hypotonic treatment, fixation, dissociation, drop piece, dye and wash
The steps such as piece, it is widely applied in many fields of biogenetics.But because the tissue of epinephelus akaara itself is special
Property, according to the method for chromosome preparation of routine, acquisition is difficult, cost is higher, it is poor to make chromosome specimen effect, time length.
The content of the invention
The technical problem to be solved in the present invention is to provide a kind of preparation method of epinephelus akaara adult fish chromosome, the present invention
Improved on the basis of the method for chromosome preparation of routine, change Hypotonic treatment time and pre-treating method, obtained
More satisfactory chromosome picture, while preparation time shortens.
The present invention is achieved by the following technical solution：
A kind of preparation method of epinephelus akaara adult fish chromosome, it is characterised in that it includes material processing early stage, prepared
Renal cell suspension, Hypotonic treatment, fixation, drop piece and dyeing；
Described material processing early stage is using phytohaemagglutinin internal injection method, by the agent of 15 μ g/g fish bodies weight
Amount, PHA is injected to the pectoral fin base portion of experiment fish, weighs the dosage injection colchicine solution of 2 μ g/g fish bodies weight after 24h by fish body,
After 2.5-3h, head-kidney is taken out；
Described Hypotonic treatment is by cell suspension 2200r/min centrifugation 5min, abandons supernatant, keep on file portion 0.8-
1ml is precipitated, and adds 7-8mL 0.75g/L KCl hypotonic mediums, and 25min, hypotonic rear four layers of gauze again mistake are handled at 30 DEG C
Filter one time.
The present invention also provides a kind of preparation method of epinephelus akaara adult fish chromosome, and it includes material processing early stage, system
Standby renal cell suspension, Hypotonic treatment, fixation, drop piece and dyeing, are comprised the following steps that：
(1) material processing early stage
Described material processing early stage is using phytohaemagglutinin internal injection method, by the agent of 15 μ g/g fish bodies weight
Amount, PHA is injected to the pectoral fin base portion of experiment fish, weighs the dosage injection colchicine solution of 2 μ g/g fish bodies weight after 24h by fish body,
After 2.5-3h, head-kidney is taken out；
(2) renal cell suspension is prepared
Head-kidney is organized in the physiological saline of 0.8% (percentage by weight) and cleaned, removes clot and other tissues, then
It is placed in the culture dish for filling 0.8% (percentage by weight) physiological saline, fully shreds, then filtered with four layers of hospital gauze into centrifugation
In pipe, 0.8% (percentage by weight) physiological saline is added, is blown and beaten several minutes with suction pipe, a moment is stood, cell suspension is made；
(3) Hypotonic treatment
By cell suspension 2200r/min centrifugation 5min, supernatant is abandoned, portion 0.8-1ml precipitations of keeping on file, adds 7-
8mL 0.75g/L KCl hypotonic mediums, 25min is handled at 30 DEG C, is filtered again one time with four layers of gauze after hypotonic；
(4) it is fixed
Filtrate 2200r/min centrifugation 5min after Hypotonic treatment filtering, remove supernatant, portion 0.8-1ml precipitations of keeping on file, add
7mL Kanos fixer, the composition of the Kano fixer are methanol and glacial acetic acid, and the volume ratio of methanol and glacial acetic acid is 3: 1, is used
Suction pipe, which is slowly blown and beaten, is precipitated to abundant mixing, and 2200r/min centrifugations 5min, abandons supernatant, keep on file portion 0.8-1ml after fixed 30min
Precipitation, adds 7mL fixers, fixed, centrifugation, repeats above-mentioned steps twice；
(5) piece is dripped
Supernatant is abandoned after 3rd fixation, adds Kano fixer described in step (4) to 1.8-2mL, gently bullet beats centrifuge tube bottom
Portion, drop piece, described heat drop piece method are carried out using heat drop piece method：Clean slide will be cleaned it is placed in 50 DEG C of incubator and preheats
More than 30min, 2 slides being taken out every time and carry out drop piece, are dripped in 0.8-1m eminence on slide, every drop 1-2 drop is natural
Air-dry；
(6) dye
After slide is completely dried, 30min is dyed with the Giemsa dye liquors of 10% (volume ratio), after done with distilled water flushing
Dried after net, micro- Microscopic observation is simultaneously taken pictures.
The beneficial effect of the present invention compared with prior art：
The present invention uses the ground method of shot phytohaemagglutinin, while increases injection concentration, by 24h
Good effect just can be obtained afterwards, saves the time.
The present invention has been hardly resulted in due to the histocyte characteristic of epinephelus akaara when carrying out Hypotonic treatment by prior art
Whole cell, therefore the change prior art of the invention Hypotonic treatment 30-40min typically under the conditions of 37 DEG C side
Method, the key technical feature of the invention Hypotonic treatment 25min i.e. under the conditions of 30 DEG C is obtained, obtains clearly chromosome map
Piece.
Brief description of the drawings
The chromosome picture obtained after the processing of Fig. 1 the inventive method
The chromosome picture obtained after 37 DEG C of Hypotonic treatment 35min of Fig. 2
The chromosome picture obtained after 37 DEG C of Hypotonic treatment 40min of Fig. 3
Embodiment
A kind of preparation method of epinephelus akaara adult fish chromosome, it include processing material early stage, prepare renal cell suspension,
Hypotonic treatment, fixation, drop piece and dyeing, are comprised the following steps that：
(1) material processing early stage
Described material processing early stage uses phytohaemagglutinin internal injection method, by the agent of 15 μ g/g fish bodies weight
Amount, PHA is injected to the pectoral fin base portion of experiment fish, by the dosage injection colchicine solution of 2 μ g/g fish bodies weight after 24h, after about 3h
Dock the 20min that loses blood, and takes out head-kidney；
(2) renal cell suspension is prepared
Head-kidney is organized in the physiological saline of 0.8% (percentage by weight) and cleaned, removes clot and other tissues, then
It is placed in the culture dish for filling 0.8% (percentage by weight) physiological saline, fully shreds, then filtered with four layers of hospital gauze into 10ml
In centrifuge tube, the physiological saline of 0.8% (percentage by weight) is added, is blown and beaten several minutes with suction pipe, a moment is stood, it is thin that 8ml is made
Born of the same parents' suspension；
(3) Hypotonic treatment
By cell suspension 2200r/min centrifugation 5min, supernatant is abandoned, portion 0.8-1ml precipitations of keeping on file, adds 7-
8mL 0.75g/L KCl hypotonic mediums, 25min is handled at 30 DEG C, is filtered again one time with four layers of gauze after hypotonic；
(4) it is fixed
Filtrate 2200r/min centrifugation 5min after Hypotonic treatment filtering, remove supernatant, portion 0.8-1ml precipitations of keeping on file, add
7mL Kanos fixer, the composition of the Kano fixer are methanol and glacial acetic acid, and the volume ratio of methanol and glacial acetic acid is 3: 1, is used
Suction pipe, which is slowly blown and beaten, is precipitated to abundant mixing, centrifuges 5min with 2200r/min after fixed 30min, abandons supernatant, keep on file portion 0.8-
1ml is precipitated, and adds 7mL fixers, fixed, centrifugation, repeats above-mentioned steps twice；
(5) piece is dripped
Supernatant is abandoned after 3rd fixation, adds Kano fixer to 1.8-2mL, drips before piece that gently bullet beats centrifugation bottom of the tube, is distinguished
Drop piece, described heat drop piece method are carried out using heat drop piece method：Clean slide will be cleaned it is placed in 50 DEG C of incubator and preheats
More than 30min, 2 slides are taken out every time and carry out drop piece, are dripped in 1m eminence on slide, every drop 1-2 drop, natural wind
It is dry；
(6) dye
Dye 30min with the Giemsa dye liquors of 10% (volume ratio) after slide is completely dried, after done with distilled water flushing
Dried after net, micro- Microscopic observation is simultaneously taken pictures as shown in Figure 1.Described Giemsa dye liquors liquid is Jim Sa stoste：Dilution (phosphorus
Acid buffer)=1:9 are made into.
While the present embodiment is carried out, early stage of the invention carries out Hypotonic treatment according to prior art, and treatment conditions are
0.75g/L KCl hypotonic mediums, 35,40min is handled respectively at 37 DEG C, the same embodiment of the present invention of other operating procedures, as a result as schemed
2nd, shown in 3.
Claims (1)
1. a kind of preparation method of epinephelus akaara adult fish chromosome, it is characterised in that it includes material processing early stage, prepares kidney
Cell suspension, Hypotonic treatment, fixation, drop piece and dyeing, are comprised the following steps that：
(1) material processing early stage
Described processing material early stage be using phytohaemagglutinin internal injection method, by the dosage of 15 μ g/g fish bodies weight,
PHA is injected to the pectoral fin base portion of experiment fish, the dosage injection colchicine solution of 2 μ g/g fish bodies weight is pressed after 24h, in 2.5-3h
Afterwards, dock the 20min that loses blood, and takes out head-kidney；
(2) renal cell suspension is prepared
Head-kidney is organized in the physiological saline of 0.8% (percentage by weight) and cleaned, clot and other tissues is removed, is subsequently placed in
In the culture dish for filling 0.8% (percentage by weight) physiological saline, fully shred, then filtered with four layers of hospital gauze into centrifuge tube
It is interior, 0.8% (percentage by weight) physiological saline is added, is blown and beaten several minutes with suction pipe, a moment is stood, cell suspension is made；
(3) Hypotonic treatment
By cell suspension 2200r/min centrifugation 5min, supernatant is abandoned, portion 0.8-1ml precipitations of keeping on file, adds 7-8mL
0.75g/L KCl hypotonic mediums, 25min is handled at 30 DEG C, is filtered again one time with four layers of gauze after hypotonic；
(4) it is fixed
Filtrate 2200r/min centrifugation 5min after Hypotonic treatment filtering, remove supernatant, portion 0.8-1ml precipitations of keeping on file, add 7mL cards
Promise fixer, the composition of the Kano fixer are methanol and glacial acetic acid, and the volume ratio of methanol and glacial acetic acid is 3: 1, uses suction pipe
Slowly piping and druming is precipitated to abundant mixing, and 2200r/min centrifuges 5min after fixed 30min, abandons supernatant, the portion 0.8-1ml of keeping on file sinks
Form sediment, add 7mL fixers, it is fixed, centrifugation, repeat above-mentioned steps twice；
(5) piece is dripped
Supernatant is abandoned after 3rd fixation, adds Kano fixer described in step (4) to 1.8-2mL, gently bullet beats centrifugation bottom of the tube,
Drop piece, described heat drop piece method are carried out using heat drop piece method：Clean slide will be cleaned it is placed in 50 DEG C of incubator and preheats
More than 30min, 2 slides are taken out every time and carry out drop piece, are dripped in 0.8-1m eminence on slide, every drop 1-2 drop, from
So air-dry；
(6) dye
After slide is completely dried, after the Giemsa dye liquors of 10% (volume ratio) dyeing 30min, with distilled water flushing it is clean after
Dry, micro- Microscopic observation is simultaneously taken pictures.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410682981.2A CN104483178B (en) | 2014-11-24 | 2014-11-24 | The preparation method of epinephelus akaara adult fish chromosome |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410682981.2A CN104483178B (en) | 2014-11-24 | 2014-11-24 | The preparation method of epinephelus akaara adult fish chromosome |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104483178A CN104483178A (en) | 2015-04-01 |
| CN104483178B true CN104483178B (en) | 2018-03-30 |
Family
ID=52757748
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410682981.2A Expired - Fee Related CN104483178B (en) | 2014-11-24 | 2014-11-24 | The preparation method of epinephelus akaara adult fish chromosome |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104483178B (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106289907A (en) * | 2016-08-11 | 2017-01-04 | 中国水产科学研究院黄海水产研究所 | Quickly prepare small-scale marine fishes or the method for larva and juvenile division phases chromosome |
| CN106680056A (en) * | 2016-12-05 | 2017-05-17 | 浙江海洋大学 | Preparation method for gill chromosome of sepiella maindroni |
| CN110713920A (en) * | 2018-07-13 | 2020-01-21 | 迈健医药科技无锡有限公司 | Filtering centrifugal tube type lymphocyte culture tube and culture method |
| CN109883797B (en) * | 2019-04-01 | 2020-03-24 | 中国水产科学研究院黄海水产研究所 | Simple method for preparing coilia mystus chromosome |
| CN112393957B (en) * | 2019-08-15 | 2023-07-07 | 杭州市农业科学研究院 | Simple preparation method of fish chromosome karyotype |
| CN115046808B (en) * | 2022-07-20 | 2023-09-01 | 浙江省淡水水产研究所 | Minimally invasive blood sampling method and chromosome slicing method for soft-shelled turtle animals |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| PT1118268E (en) * | 2000-01-14 | 2002-09-30 | Exelixis Deutschland Gmbh | METHOD FOR THE CRIOPRESERVATION OF PEEZ-ZEBRA SPERM |
| CN101825532B (en) * | 2009-12-31 | 2011-12-28 | 中国水产科学研究院长江水产研究所 | Fish embryo chromosome flaking method |
| CN102509504B (en) * | 2011-09-22 | 2013-07-10 | 中国水产科学研究院黄海水产研究所 | Method for preparing chromosome specimen with sepia esculenta embryonic cell |
-
2014
- 2014-11-24 CN CN201410682981.2A patent/CN104483178B/en not_active Expired - Fee Related
Non-Patent Citations (5)
| Title |
|---|
| 4 种石斑鱼染色体核型研究;郑莲，刘楚吾，李长玲;《海洋科学》;20050430;第29卷(第4期);第51-52页第"1 材料与方法"节 * |
| 七带石斑鱼染色体核型研究;钟声平等;《中国水产科学》;20100131;第17卷(第1期);第150页第"1.2.1染色体标本的制备"节 * |
| 两种鱼类染色体制片方法的比较研究;张伟明等;《水利渔业》;20031231;第23卷(第5期);第9页第"1.2方法"节 * |
| 快速获得大量鱼类肾细胞中期分裂相的PHA体内注射法;林义浩;《水产学报》;19820930;第6卷(第3期);第202页第"材料与方法"节 * |
| 海南野生三斑石斑鱼染色体核型分析;蔡岩等;《热带生物学报》;20110630;第2卷(第2期);第98页第"1.2.1染色体标本的制备"节 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104483178A (en) | 2015-04-01 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104483178B (en) | The preparation method of epinephelus akaara adult fish chromosome | |
| RU2600871C2 (en) | Device for centrifugal filtration and cells separation system with such device for centrifugal filtration | |
| CN101596327B (en) | Method for preparing three-dimensional silk fibroin porous scaffold material | |
| CN108129547B (en) | Method for extracting extracellular polymeric substance of zoogloea | |
| CN106687152A (en) | Human liver scaffolds | |
| US11452744B2 (en) | Liquid amnion transplant product | |
| CN111084904B (en) | Cell removing method of tissue engineering scaffold | |
| CN113388575A (en) | Preparation method of mesenchymal stem cell exosome for skin injury repair | |
| CN110055213A (en) | A kind of separation method of dwarf clam egg membrane | |
| CN107164540A (en) | The quick determination method of Listeria Monocytogenes in one seed oyster | |
| CN103114074B (en) | Method for building cGVHD (chronic graft-versus-host disease) model of mouse allogene after bone marrow transplantation | |
| CN106644644A (en) | Quick and low-toxic mosquito paraffin section manufacturing method | |
| CN107412865A (en) | The decellularization kidney biological support and preparation method with anticoagulant functions of high intensity | |
| CN105586307A (en) | Separating method and culturing method for hepatic stellate cells of Mongolian gerbil | |
| CN112393957B (en) | Simple preparation method of fish chromosome karyotype | |
| Bonciog et al. | Software Validation For Automatic Heart Decellularization | |
| CN111330076B (en) | Cell removing device of tissue engineering bracket | |
| CN107219104A (en) | A kind of preparation method of Procambius clarkii chromosome | |
| CN104357380A (en) | Separation method of human epidermal stem cells | |
| CN206033773U (en) | Prokaryotes protein pretreatment systems | |
| CN108865976B (en) | Method and reagent for discretely drawing and collecting exosomes (exosomes) | |
| CN217895641U (en) | Auxiliary device for obtaining complete organoid tissues | |
| CN108572101A (en) | liquid-phase molecular sieve | |
| CN102766692A (en) | Preparation method of siganus guttatus chromosome | |
| CN208136224U (en) | A kind of fat stem cell bag for extracting |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20180330 Termination date: 20191124 |