CN104483178A - Preparation method of chromosomes of adult epinephelus akaara - Google Patents
Preparation method of chromosomes of adult epinephelus akaara Download PDFInfo
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- CN104483178A CN104483178A CN201410682981.2A CN201410682981A CN104483178A CN 104483178 A CN104483178 A CN 104483178A CN 201410682981 A CN201410682981 A CN 201410682981A CN 104483178 A CN104483178 A CN 104483178A
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Abstract
The invention discloses a preparation method of chromosomes of adult epinephelus akaara and belongs to the field of cell biology. The preparation method comprises the following steps: pretreatment of materials, preparation of kidney cell suspension, low-permeation treatment, fixation, dropping and dyeing, wherein the pretreatment of the materials comprises the steps of with an in vivo injection method of phytohemagglutinin (PHA), injecting PHA into the pectoral-fin base part of the experimental adult epinephelus akaara with 15 micrograms in each gram of the adult epinephelus akaara; after 24 hours, injecting colchicine solution with 2 micrograms in each gram of the adult epinephelus akaara; and after 2.5-3 hours, taking out the head kidney. The preparation method disclosed by the invention has the advantages that the method for one-time injection of PHA is adopted, simultaneously, the injection concentration is increased, and good effect can be obtained after 24 hours, so that the time is saved; and due to the characteristics of tissue cells of the epinephelus akaara, complete cells are not easily obtained when the low-permeation treatment is carried out in the prior art, so that the prior art is creatively changed and a clear chromosome image is obtained.
Description
Technical field
The invention belongs to cell biology, relate to a kind of red chromosomal preparation method of lithosporic adult fish particularly.
Background technology
The preparation manipulation of chromosome specimen is simple, cheap, experimental site requires low feature, it is traditional biogenetics investigative technique always, chromosome specimen preparation comprises: pre-treatment, Hypotonic treatment, fix, dissociate, drip sheet, the step such as dye and develop a film, be widely applied in many fields of biogenetics.But due to the tissue characteristics of epinephelus akaara self, method of chromosome preparation conveniently, obtains difficulty, cost is higher, to make chromosome specimen weak effect, time long.
Summary of the invention
The technical problem to be solved in the present invention is to provide the chromosomal preparation method of a kind of epinephelus akaara adult fish, the present invention improves on the basis of the method for chromosome preparation of routine, change Hypotonic treatment time and pre-treating method, obtain more satisfactory chromosome picture, preparation time shortens simultaneously.
The present invention is achieved by the following technical solution:
The chromosomal preparation method of a kind of epinephelus akaara adult fish, is characterized in that it comprises material and in earlier stage processes, prepares renal cell suspension, Hypotonic treatment, fixing, droplet sheet and dyeing;
Described material is treated to injection in employing phytohaemagglutinin body early stage, by the dosage of 15 μ g/g fish body weight, by the dosage injection colchicine solution of fish body weight 2 μ g/g fish body weight after the injection of pectoral fin base portion PHA, the 24h of experiment fish, after 2.5-3h, take out head-kidney;
Described Hypotonic treatment is by the centrifugation 5min of cell suspension 2200r/min, abandons supernatant, and the portion 0.8-1ml that keeps on file precipitates, and adds the KCl hypotonic medium of the 0.75g/L of 7-8mL, processes 25min at 30 DEG C, and hypotonic rear use four layers of gauze filter one time again.
The present invention also provides a kind of epinephelus akaara adult fish chromosomal preparation method, and it comprises material and in earlier stage processes, prepares renal cell suspension, Hypotonic treatment, fixing, droplet sheet and dyeing, and concrete steps are as follows:
(1) material process in early stage
Described material is treated to injection in employing phytohaemagglutinin body early stage, by the dosage of 15 μ g/g fish body weight, by the dosage injection colchicine solution of fish body weight 2 μ g/g fish body weight after the injection of pectoral fin base portion PHA, the 24h of experiment fish, after 2.5-3h, take out head-kidney;
(2) renal cell suspension is prepared
Head-kidney is organized in the physiological saline of 0.8% (percentage by weight) and cleans, removing clot and other tissue, then the double dish filling 0.8% (percentage by weight) physiological saline is placed in, fully shred, filter in centrifuge tube with four layers of hospital gauze again, add 0.8% (percentage by weight) physiological saline, blow and beat several minutes with suction pipe, leave standstill a moment, make cell suspension;
(3) Hypotonic treatment
By the centrifugation 5min of cell suspension 2200r/min, abandon supernatant, the portion 0.8-1ml that keeps on file precipitates, and adds the KCl hypotonic medium of the 0.75g/L of 7-8mL, processes 25min at 30 DEG C, and hypotonic rear use four layers of gauze filter one time again;
(4) fixing
The centrifugal 5min of filtrate 2200r/min after Hypotonic treatment filters, remove supernatant, the portion 0.8-1ml that keeps on file precipitates, add 7mL Kano immobile liquid, the composition of described Kano immobile liquid is methyl alcohol and glacial acetic acid, and the volume ratio of methyl alcohol and glacial acetic acid is 3: 1, slowly blow and beat with suction pipe and be precipitated to abundant mixing, the centrifugal 5min of 2200r/min after fixing 30min, abandon supernatant, the portion 0.8-1ml that keeps on file precipitates, add 7mL immobile liquid, fixing, centrifugal, then repeat above-mentioned steps twice;
(5) sheet is dripped
Supernatant is abandoned after fixing for 3rd time, add Kano immobile liquid described in step (4) to 1.8-2mL, bullet is beaten bottom centrifuge tube gently, adopt heat to drip sheet method to carry out dripping sheet, described heat drips sheet method: clean clean slide is placed on more than preheating 30min in the incubator of 50 DEG C, and each taking-up 2 slides carry out dripping a sheet, and the eminence in 0.8-1m drips on microslide, every sheet drips 1-2 and drips, natural air drying;
(6) dye
After slide bone dry, with the Giemsa dye liquor dyeing 30min of 10% (volume ratio), dry after rear distilled water flushing is clean, basis of microscopic observation is also taken pictures.
The present invention's beneficial effect compared with prior art:
The present invention adopts the ground method of shot phytohaemagglutinin, increases injection concentration simultaneously, just obtaining good effect after 24h, saves the time.
The present invention is due to the histocyte characteristic of epinephelus akaara, be difficult to when carrying out Hypotonic treatment by prior art obtain complete cell, therefore the change prior art of the invention generally method of Hypotonic treatment 30-40min under 37 DEG C of conditions, obtain key technical feature of the present invention i.e. Hypotonic treatment 25min under 30 DEG C of conditions, obtain chromosome picture clearly.
Accompanying drawing explanation
The chromosome picture obtained after the process of Fig. 1 the inventive method
The chromosome picture obtained after Fig. 2 37 DEG C of Hypotonic treatment 35min
The chromosome picture obtained after Fig. 3 37 DEG C of Hypotonic treatment 40min
Embodiment
The chromosomal preparation method of a kind of epinephelus akaara adult fish, it comprises material and in earlier stage processes, prepares renal cell suspension, Hypotonic treatment, fixing, droplet sheet and dyeing, and concrete steps are as follows:
(1) material process in early stage
Described material process in early stage adopts injection in phytohaemagglutinin body, by the dosage of 15 μ g/g fish body weight, by the dosage injection colchicine solution of 2 μ g/g fish body weight after the injection of pectoral fin base portion PHA, the 24h of experiment fish, dock after about 3h 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 cleans, removing clot and other tissue, then the double dish filling 0.8% (percentage by weight) physiological saline is placed in, fully shred, filter in 10ml centrifuge tube with four layers of hospital gauze again, add the physiological saline of 0.8% (percentage by weight), blow and beat several minutes with suction pipe, leave standstill a moment, make 8ml cell suspension;
(3) Hypotonic treatment
By the centrifugation 5min of cell suspension 2200r/min, abandon supernatant, the portion 0.8-1ml that keeps on file precipitates, and adds the KCl hypotonic medium of the 0.75g/L of 7-8mL, processes 25min at 30 DEG C, and hypotonic rear use four layers of gauze filter one time again;
(4) fixing
The centrifugal 5min of filtrate 2200r/min after Hypotonic treatment filters, remove supernatant, the portion 0.8-1ml that keeps on file precipitates, add 7mL Kano immobile liquid, the composition of described Kano immobile liquid is methyl alcohol and glacial acetic acid, and the volume ratio of methyl alcohol and glacial acetic acid is 3: 1, slowly blow and beat with suction pipe and be precipitated to abundant mixing, with the centrifugal 5min of 2200r/min after fixing 30min, abandon supernatant, the portion 0.8-1ml that keeps on file precipitates, add 7mL immobile liquid, fixing, centrifugal, then repeat above-mentioned steps twice;
(5) sheet is dripped
Supernatant is abandoned after fixing for 3rd time, add Kano immobile liquid to 1.8-2mL, before dripping sheet, bullet is beaten bottom centrifuge tube gently, adopt heat to drip sheet method respectively to carry out dripping sheet, described heat drips sheet method: clean clean slide is placed on more than preheating 30min in the incubator of 50 DEG C, and each taking-up 2 slides carry out dripping a sheet, and the eminence in 1m drips on microslide, every sheet drips 1-2 and drips, natural air drying;
(6) dye
With the Giemsa dye liquor dyeing 30min of 10% (volume ratio) after slide bone dry, dry after rear distilled water flushing is clean, basis of microscopic observation is also taken pictures as shown in Figure 1.Described Giemsa dye liquor liquid is Jim Sa stoste: dilution (phosphate buffer)=1:9 is made into.
While carrying out the present embodiment, the present invention conventionally carries out Hypotonic treatment early stage, and treatment conditions are the KCl hypotonic medium of 0.75g/L, processes 35 respectively, 40min at 37 DEG C, and the same embodiment of the present invention of other operation stepss, result as shown in Figure 2,3.
Claims (2)
1. the chromosomal preparation method of epinephelus akaara adult fish, is characterized in that it comprises material and in earlier stage processes, prepares renal cell suspension, Hypotonic treatment, fixing, droplet sheet and dyeing;
Described material is treated to injection in employing phytohaemagglutinin body early stage, by the dosage of 15 μ g/g fish body weight, by the dosage injection colchicine solution of 2 μ g/g fish body weight after the injection of pectoral fin base portion PHA, the 24h of experiment fish, after 2.5-3h, take out head-kidney;
Described Hypotonic treatment is by the centrifugation 5min of cell suspension 2200r/min, abandons supernatant, and the portion 0.8-1ml that keeps on file precipitates, and adds the KCl hypotonic medium of the 0.75g/L of 7-8mL, processes 25min at 30 DEG C, and hypotonic rear use four layers of gauze filter one time again.
2. the chromosomal preparation method of a kind of epinephelus akaara adult fish according to claim 1, it is characterized in that it comprises material and in earlier stage processes, prepares renal cell suspension, Hypotonic treatment, fixing, droplet sheet and dyeing, concrete steps are as follows:
(1) material process in early stage
Described material is treated to injection in employing phytohaemagglutinin body early stage, by the dosage of 15 μ g/g fish body weight, by the dosage injection colchicine solution of 2 μ g/g fish body weight after the injection of pectoral fin base portion PHA, the 24h of experiment fish, after 2.5-3h, take out head-kidney;
(2) renal cell suspension is prepared
Head-kidney is organized in the physiological saline of 0.8% (percentage by weight) and cleans, removing clot and other tissue, then the double dish filling 0.8% (percentage by weight) physiological saline is placed in, fully shred, filter in centrifuge tube with four layers of hospital gauze again, add 0.8% (percentage by weight) physiological saline, blow and beat several minutes with suction pipe, leave standstill a moment, make cell suspension;
(3) Hypotonic treatment
By the centrifugation 5min of cell suspension 2200r/min, abandon supernatant, the portion 0.8-1ml that keeps on file precipitates, and adds the KCl hypotonic medium of the 0.75g/L of 7-8mL, processes 25min at 30 DEG C, and hypotonic rear use four layers of gauze filter one time again;
(4) fixing
The centrifugal 5min of filtrate 2200r/min after Hypotonic treatment filters, remove supernatant, the portion 0.8-1ml that keeps on file precipitates, add 7mL Kano immobile liquid, the composition of described Kano immobile liquid is methyl alcohol and glacial acetic acid, and the volume ratio of methyl alcohol and glacial acetic acid is 3: 1, slowly blow and beat with suction pipe and be precipitated to abundant mixing, the centrifugal 5min of 2200r/min after fixing 30min, abandon supernatant, the portion 0.8-1ml that keeps on file precipitates, add 7mL immobile liquid, fixing, centrifugal, then repeat above-mentioned steps twice;
(5) sheet is dripped
Supernatant is abandoned after fixing for 3rd time, add Kano immobile liquid described in step (4) to 1.8-2mL, bullet is beaten bottom centrifuge tube gently, adopt heat to drip sheet method to carry out dripping sheet, described heat drips sheet method: clean clean slide is placed on more than preheating 30min in the incubator of 50 DEG C, and each taking-up 2 slides carry out dripping a sheet, and the eminence in 0.8-1m drips on microslide, every sheet drips 1-2 and drips, natural air drying;
(6) dye
After slide bone dry, with the Giemsa dye liquor dyeing 30min of 10% (volume ratio), dry after rear distilled water flushing is clean, basis of microscopic observation is also taken pictures.
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Cited By (6)
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| 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 |
| CN109883797A (en) * | 2019-04-01 | 2019-06-14 | 中国水产科学研究院黄海水产研究所 | A kind of method of easy long-tail anchovy chromosome preparation |
| 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 |
| CN112393957A (en) * | 2019-08-15 | 2021-02-23 | 杭州市农业科学研究院 | Simple preparation method of fish chromosome karyotype |
| CN115046808A (en) * | 2022-07-20 | 2022-09-13 | 浙江省淡水水产研究所 | Minimally invasive blood extraction method and chromosome flaking method for turtle animals |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 |
| CN109883797A (en) * | 2019-04-01 | 2019-06-14 | 中国水产科学研究院黄海水产研究所 | A kind of method of easy long-tail anchovy chromosome preparation |
| CN112393957A (en) * | 2019-08-15 | 2021-02-23 | 杭州市农业科学研究院 | Simple preparation method of fish chromosome karyotype |
| CN115046808A (en) * | 2022-07-20 | 2022-09-13 | 浙江省淡水水产研究所 | Minimally invasive blood extraction method and chromosome flaking method for turtle animals |
| 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 |
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