CN112393957A - Simple preparation method of fish chromosome karyotype - Google Patents
Simple preparation method of fish chromosome karyotype Download PDFInfo
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- CN112393957A CN112393957A CN201910755030.6A CN201910755030A CN112393957A CN 112393957 A CN112393957 A CN 112393957A CN 201910755030 A CN201910755030 A CN 201910755030A CN 112393957 A CN112393957 A CN 112393957A
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- 241000251468 Actinopterygii Species 0.000 title claims abstract description 57
- 210000000349 chromosome Anatomy 0.000 title claims abstract description 33
- 238000002360 preparation method Methods 0.000 title claims abstract description 25
- 239000011521 glass Substances 0.000 claims abstract description 42
- 210000004027 cell Anatomy 0.000 claims abstract description 35
- IAKHMKGGTNLKSZ-INIZCTEOSA-N (S)-colchicine Chemical compound C1([C@@H](NC(C)=O)CC2)=CC(=O)C(OC)=CC=C1C1=C2C=C(OC)C(OC)=C1OC IAKHMKGGTNLKSZ-INIZCTEOSA-N 0.000 claims abstract description 30
- 239000006285 cell suspension Substances 0.000 claims abstract description 26
- 210000005084 renal tissue Anatomy 0.000 claims abstract description 23
- 238000004043 dyeing Methods 0.000 claims abstract description 21
- 239000000815 hypotonic solution Substances 0.000 claims abstract description 20
- 238000002347 injection Methods 0.000 claims abstract description 16
- 239000007924 injection Substances 0.000 claims abstract description 16
- 229960001338 colchicine Drugs 0.000 claims abstract description 15
- 210000003292 kidney cell Anatomy 0.000 claims abstract description 13
- 238000004140 cleaning Methods 0.000 claims abstract description 7
- 239000000463 material Substances 0.000 claims abstract description 7
- 239000000243 solution Substances 0.000 claims description 30
- 238000000034 method Methods 0.000 claims description 26
- 101710154606 Hemagglutinin Proteins 0.000 claims description 21
- 101710093908 Outer capsid protein VP4 Proteins 0.000 claims description 21
- 101710135467 Outer capsid protein sigma-1 Proteins 0.000 claims description 21
- 101710176177 Protein A56 Proteins 0.000 claims description 21
- 239000000185 hemagglutinin Substances 0.000 claims description 21
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- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 15
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 10
- 210000003734 kidney Anatomy 0.000 claims description 8
- 210000000006 pectoral fin Anatomy 0.000 claims description 8
- 238000007664 blowing Methods 0.000 claims description 7
- 238000001035 drying Methods 0.000 claims description 7
- 238000001727 in vivo Methods 0.000 claims description 7
- 238000005406 washing Methods 0.000 claims description 7
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- 210000001519 tissue Anatomy 0.000 claims description 6
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- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 5
- 229960000583 acetic acid Drugs 0.000 claims description 5
- 238000011010 flushing procedure Methods 0.000 claims description 5
- 239000012362 glacial acetic acid Substances 0.000 claims description 5
- 230000007547 defect Effects 0.000 abstract description 3
- 238000002203 pretreatment Methods 0.000 abstract 1
- 239000000725 suspension Substances 0.000 abstract 1
- 239000012981 Hank's balanced salt solution Substances 0.000 description 8
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Images
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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
- G01N1/2813—Producing thin layers of samples on a substrate, e.g. smearing, spinning-on
-
- 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
Abstract
A simple preparation method of fish chromosome karyotype comprises pre-treatment of material; preparing a suspension of kidney cells; performing hypotonic treatment; and (3) fixing and dyeing. The invention overcomes the defects of the prior art, the application prepares the renal cell suspension by directly using a cell screen in KCl hypotonic solution after cleaning the head and kidney tissues, and then directly performs hypotonic and fixed treatment on a glass slide; the purpose of doing so is to avoid centrifugal operation, so that the preparation does not depend on a centrifugal machine to centrifugally collect cells, thereby avoiding the influence of the centrifugal operation on the cells and improving the success rate of preparing the nuclear type slide specimen; in addition, PHA and colchicine can be injected simultaneously in the application, and a fish chromosome karyotype slide specimen can still be prepared; therefore, the waiting time between PHA and colchicine injection is reduced, and the preparation time of the fish chromosome karyotype slide specimen is shortened.
Description
Technical Field
The invention relates to the technical field of cell biology, in particular to a simple preparation method of a fish chromosome karyotype.
Background
The preparation of the chromosome specimen is simple in operation, low in price and low in requirement of an experimental site, and is always a traditional biological genetics research technology, and the preparation of the chromosome specimen comprises the following steps: pretreatment, hypotonic treatment, fixation, dripping, dyeing, washing and the like are widely applied to many fields of biological genetics.
According to the published standard of fish chromosome preparation, the conventional methods for preparing fish chromosomes generally comprise 3 types: somatic cell in vitro culture method, somatic cell in vivo culture method, somatic cell direct method, and embryonic cell direct method, which are mainly used for preparing chromosome karyotype of embryonic cells. However, according to the conventional chromosome preparation method, the acquisition is difficult, the cost is high, the effect of preparing a chromosome specimen is poor, the time is long, and the dependence on equipment and experimental conditions are high.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a simple preparation method of the fish chromosome karyotype, which overcomes the defects of the prior art, the fastest required time for preparing the fish chromosome karyotype is only 3-4 hours, and the preparation efficiency of the fish chromosome karyotype is improved; in addition, the method does not use a centrifuge to separate cells, reduces the dependence of the experiment on the instrument, avoids the influence of centrifugal operation on the cells, and improves the success rate of preparing the nuclear type slide specimen.
In order to achieve the purpose, the invention is realized by the following technical scheme:
a simple preparation method of a fish chromosome karyotype comprises the following steps:
(1) performing preliminary treatment on the material, namely injecting plant hemagglutinin to the basal part of the pectoral fin of the experimental fish according to the dose of 10-30 mu g/g fish weight, then injecting colchicine solution according to the dose of 1-2 mu g/g fish weight, taking out the head and kidney after 1-2h, and placing the head and kidney in Hank's solution or normal saline for later use;
(2) preparing a kidney cell suspension, namely cleaning the head and kidney tissues taken out in the step (1) in Hank's solution or physiological saline to remove blood clots and other tissues, then placing the head and kidney tissues in a cell screen, placing the cell screen in a culture dish containing KCl hypotonic solution, repeatedly tearing the head and kidney tissues placed in the cell screen, taking out the cell screen, blowing the hypotonic solution containing the head and kidney cells for 1-2 minutes, and standing to prepare the cell suspension;
(3) performing hypotonic treatment, namely taking 10-15 culture dishes, placing 1 glass slide in each culture dish, dropwise adding the cell suspension prepared in the step (2) onto the glass slide in the culture dish, adding 1ml of KCl hypotonic solution onto the glass slide, covering a culture dish cover, and performing hypotonic treatment for 30-40 minutes at 30 ℃;
(4) fixing, namely after hypotonic treatment is finished, adding 5-6mL of freshly prepared Carnoy fixing liquid into the culture dish for fixing for 25-35 minutes, replacing the freshly prepared Carnoy fixing liquid, fixing for 1-2 hours, taking out the glass slide, flushing the glass slide for 2-3 times by using the freshly prepared Carnoy fixing liquid, and then placing the glass slide in the air for drying;
(5) and (3) dyeing, namely after the slide is completely dried, carrying out dyeing treatment for 5-10 minutes, then washing the slide clean with distilled water, and airing to obtain the fish chromosome karyotype.
Preferably, the plant hemagglutinin is injected into the basal part of the pectoral fin of the experimental fish by using the in vivo injection method of the plant hemagglutinin in the step (1).
Preferably, in the step (1), the plant hemagglutinin may be injected 1 time before the injection of the plant hemagglutinin 15 to 20 hours, and the colchicine solution may be injected at the same time or 1 to 2 hours after the injection of the plant hemagglutinin for the second time.
Preferably, in the step (1), the colchicine solution is injected 4-6h after the injection of the plant hemagglutinin.
Preferably, in the step (2), the mesh opening of the cell screen is 100 μm.
Preferably, the concentration of KCl hypotonic solution in the step (2) and the step (3) is 0.75 g/L.
Preferably, in the step (3), the cell suspension is aspirated by a dropper, and the cell suspension is dripped from a height of 20-30 cm onto glass slides placed in a culture dish, and 3 drops are dripped on each glass slide.
Preferably, in the step (4), the Carnot's fixative is added to the culture dish in an amount to submerge the slide glass in the culture dish.
Preferably, in the step (4), the volume ratio of methanol to glacial acetic acid in the Carnot's stationary solution is 3: 1.
Preferably, in the step (5), the dyeing treatment is performed by using a giemsa dyeing solution with a volume ratio of 10%.
The invention provides a simple preparation method of a fish chromosome karyotype. The method has the following beneficial effects: the preparation method has the advantages of short preparation time, few preparation steps and simple operation, the fastest time for preparing the fish chromosome karyotype is only 3-4 hours, and the preparation efficiency of the fish chromosome karyotype is improved; in addition, the method does not use a centrifuge to separate cells, reduces the dependence of the experiment on the instrument, avoids the influence of centrifugal operation on the cells, and improves the success rate of preparing the nuclear type slide specimen.
Drawings
In order to more clearly illustrate the present invention or the prior art solutions, the drawings that are needed in the description of the prior art will be briefly described below.
FIG. 1 is a schematic diagram of the method steps of the present invention;
FIG. 2 is a chromosome map of triangular bream observed under a 40-fold objective using the method of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described clearly and completely with reference to the accompanying drawings.
Example one
As shown in FIG. 1, a simple method for preparing a fish karyotype comprises the following steps:
(1) the material is processed in an earlier stage, firstly, according to the dosage of 25 mug/g fish weight, the plant hemagglutinin is injected in vivo to the basal part of the pectoral fin of the experimental fish, and simultaneously, the colchicine solution is injected according to the dosage of 2 mug/g fish weight, and after 2 hours, the head and kidney are taken out and placed in Hank's solution or normal saline for standby;
(2) preparing a kidney cell suspension, namely cleaning the head kidney tissue taken out in the step (1) in Hank's solution or physiological saline, removing blood clots and other tissues, then placing the head kidney tissue in a cell screen with the screen hole diameter of 100 mu m, placing the cell screen in a culture dish containing 0.75g/L KCl hypotonic solution, repeatedly tearing the head kidney tissue placed in the cell screen, taking out the cell screen, blowing the hypotonic solution containing the head kidney cells for 1 minute, and standing to prepare the cell suspension;
(3) performing hypotonic treatment, namely taking 10 culture dishes, placing 1 glass slide in each culture dish, sucking the cell suspension prepared in the step (2) through a dropper, dripping the cell suspension onto the glass slide placed in the culture dish from a height of 20 cm, adding 1ml of 0.75g/L KCl hypotonic solution onto the glass slide, covering a culture dish cover, and performing hypotonic treatment for 40 minutes at 30 ℃;
(4) fixing, namely after the hypotonic treatment is finished, adding a freshly prepared Carnoy fixing solution into the culture dish for fixing for 30 minutes, replacing the freshly prepared Carnoy fixing solution, fixing for 1 hour, taking out the glass slide, flushing the glass slide for 3 times by using the freshly prepared Carnoy fixing solution, and then placing the glass slide in the air for drying; wherein the volume ratio of methanol to glacial acetic acid in the Carnot's stationary liquid is 3: 1, and the added Carnot's stationary liquid is based on submerging the glass slide in the culture dish;
(5) and (3) dyeing, namely after the slide is completely dried, dyeing for 5 minutes by adopting 10 volume percent Giemsa (Giemsa) dyeing solution, washing with distilled water, and airing to obtain the fish chromosome karyotype.
Example two
A simple preparation method of a fish chromosome karyotype comprises the following steps:
(1) the material is processed in the early stage, firstly, according to the dosage of 25 mug/g fish weight, the plant hemagglutinine in vivo injection method is adopted to inject the first Plant Hemagglutinine (PHA) to the base of the pectoral fin of the experimental fish, then the second Plant Hemagglutinine (PHA) is injected after 15h, and simultaneously colchicine solution is injected according to the dosage of 2 mug/g fish weight, and the head and kidney are taken out after 2h and placed in Hank's solution or normal saline for standby; the number of metaphase cells was increased by injecting the plant hemagglutinin in two separate injections.
(2) Preparing a kidney cell suspension, namely cleaning the head kidney tissue taken out in the step (1) in Hank's solution or physiological saline, removing blood clots and other tissues, then placing the head kidney tissue in a cell screen with the screen hole diameter of 100 mu m, placing the cell screen in a culture dish containing 0.75g/L KCl hypotonic solution, repeatedly tearing the head kidney tissue placed in the cell screen, taking out the cell screen, blowing the hypotonic solution containing the head kidney cells for 2 minutes, and standing to prepare the cell suspension;
(3) performing hypotonic treatment, namely taking 10 culture dishes, placing 1 glass slide in each culture dish, sucking the cell suspension prepared in the step (2) through a dropper, dripping the cell suspension onto the glass slide placed in the culture dish from a height of 20-30 cm, adding 1ml of 0.75g/L KCl hypotonic solution onto the glass slide, covering a culture dish cover, and performing hypotonic treatment for 30 minutes at 30 ℃;
(4) fixing, namely after hypotonic treatment is finished, adding 5-6mL of freshly prepared Carnoy fixing liquid into the culture dish for fixing for 35 minutes, replacing the freshly prepared Carnoy fixing liquid, fixing for 1.5 hours, taking out the glass slide, flushing the glass slide for 3 times by using the freshly prepared Carnoy fixing liquid, and then placing the glass slide in the air for drying; wherein the volume ratio of methanol to glacial acetic acid in the Carnot's stationary liquid is 3: 1, and the added Carnot's stationary liquid is based on submerging the glass slide in the culture dish;
(5) and (3) dyeing, namely after the slide is completely dried, dyeing for 7 minutes by adopting 10 volume percent Giemsa (Giemsa) dyeing solution, washing with distilled water, and airing to obtain the fish chromosome karyotype.
EXAMPLE III
A simple preparation method of a fish chromosome karyotype comprises the following steps:
(1) the material is processed in an earlier stage, firstly, according to the dosage of 25 mug/g fish weight, the plant hemagglutinin is injected to the basal part of the pectoral fin of the experimental fish by adopting the plant hemagglutinin in vivo injection method, then the colchicine solution is injected according to the dosage of 2 mug/g fish weight, the interval time between the injection of the Plant Hemagglutinin (PHA) and the colchicine solution is 4 hours, and after 2 hours, the head and kidney are taken out and placed in Hank's solution or normal saline for standby; the number of metaphase cells was increased by injecting the plant hemagglutinin in two separate injections.
(2) Preparing a kidney cell suspension, namely cleaning the head kidney tissue taken out in the step (1) in Hank's solution or physiological saline, removing blood clots and other tissues, then placing the head kidney tissue in a cell screen with the screen hole diameter of 100 mu m, placing the cell screen in a culture dish containing 0.75g/L KCl hypotonic solution, repeatedly tearing the head kidney tissue placed in the cell screen, taking out the cell screen, blowing the hypotonic solution containing the head kidney cells for 2 minutes, and standing to prepare the cell suspension;
(3) performing hypotonic treatment, namely taking 10 culture dishes, placing 1 glass slide in each culture dish, sucking the cell suspension prepared in the step (2) through a dropper, dripping the cell suspension onto the glass slide placed in the culture dish from a height of 30 cm, adding 1ml of 0.75g/L KCl hypotonic solution onto the glass slide, covering a culture dish cover, and performing hypotonic treatment for 30 minutes at 30 ℃;
(4) fixing, namely after hypotonic treatment is finished, adding 5-6mL of freshly prepared Carnoy fixing liquid into the culture dish for fixing for 35 minutes, replacing the freshly prepared Carnoy fixing liquid, fixing for 1.5 hours, taking out the glass slide, flushing the glass slide for 3 times by using the freshly prepared Carnoy fixing liquid, and then placing the glass slide in the air for drying; wherein the volume ratio of methanol to glacial acetic acid in the Carnot's stationary liquid is 3: 1, and the added Carnot's stationary liquid is based on submerging the glass slide in the culture dish;
(5) and (3) dyeing, namely after the slide is completely dried, dyeing for 7 minutes by adopting 10 volume percent Giemsa (Giemsa) dyeing solution, washing with distilled water, and airing to obtain the fish chromosome karyotype.
Comparative example
The preparation method of the fish chromosome karyotype in the prior art comprises the following steps:
(1) performing early-stage treatment on the material, injecting PHA (the injection dose is generally 10-30 mu g/g of the weight of the fish) to the basal part of the pectoral fin of the experimental fish by adopting a plant hemagglutinin in-vivo injection method, injecting a colchicine solution (the injection dose is generally 1-2 mu g/g of the weight of the fish) after 24 hours (generally within the range of 6 hours-120 hours), and taking out the head and kidney after 5 hours (generally within the range of 2 hours-6 hours);
(2) preparing a kidney cell suspension, cleaning head and kidney tissues in 0.8 weight percent of physiological saline, removing blood clots and other tissues, then placing the head and kidney tissues in a culture dish containing the physiological saline, fully shearing the head and kidney tissues, filtering the head and kidney tissues in a centrifugal tube by using four layers of medical gauze, adding the physiological saline, blowing the head and kidney tissues for several minutes by using a straw, and standing the head and kidney tissues for a moment to prepare a cell suspension;
(3) performing hypotonic treatment, namely centrifuging the cell suspension for 5min at the speed of 2000r/min (generally within the range of 500-3000 r/min), discarding the supernatant, leaving 0.8-1mL of sediment at the bottom, adding 10mL (generally within the range of 3-10 mL) of 0.75g/L KCl hypotonic solution, treating for 30min at 30 ℃, and filtering again by using four layers of gauze after hypotonic treatment;
(4) fixing, centrifuging the filtered filtrate at 2000r/min for 5 minutes after hypotonic treatment, removing the supernatant, leaving 0.8-1mL of sediment at the bottom, adding 10mL (generally within the range of 2-10 mL) of Carnot fixing solution, slowly blowing and beating the sediment by using a suction pipe until the sediment is fully and uniformly mixed, centrifuging at 2000r/min for 5 minutes after fixing for more than 10 minutes, discarding the supernatant, leaving 0.8-1mL of sediment at the bottom, adding 7mL of fixing solution, fixing, centrifuging, and repeating the steps twice;
(5) dropping the slide, namely removing the supernatant after 3 rd fixation, adding Carnot fixing liquid to 1mL, slightly flicking the bottom of the centrifuge tube, dropping the slide by adopting a hot-drop method or a freezing-drop method (the hot-drop method means that a clean slide is placed in an incubator at 50 ℃ and preheated for more than 30min for standby, the freezing-drop method means that the clean slide is placed in an incubator at-20 ℃ and pre-frozen for more than 30min for standby), taking out 2 slides each time for dropping the slide, dropping the slide on a glass slide at a high position of 0.5-1m, dropping 1-2 drops on each slide, and naturally drying;
(6) dyeing, after the slide is completely dried, dyeing for 30min by using 10% (volume ratio) Giemsa dye solution, washing with distilled water, and drying to obtain the fish chromosome karyotype.
According to the comparison of the preparation methods, the following steps can be obtained: after the head and kidney tissues are cleaned, a cell screen is directly used in KCl hypotonic solution to prepare a kidney cell suspension, and then hypotonic and fixed treatment is directly carried out on a glass slide; the purpose of doing so is to avoid centrifugal operation, so that the preparation does not depend on a centrifugal machine to centrifugally collect cells, thereby avoiding the influence of the centrifugal operation on the cells and improving the success rate of preparing the nuclear type slide specimen; in addition, PHA and colchicine can be injected simultaneously in the application, and a fish chromosome karyotype slide specimen can still be prepared; therefore, the waiting time between PHA and colchicine injection is reduced, and the preparation time of the fish chromosome karyotype slide specimen is shortened.
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Claims (10)
1. A simple preparation method of a fish chromosome karyotype is characterized by comprising the following steps:
(1) performing preliminary treatment on the material, namely injecting plant hemagglutinin to the basal part of the pectoral fin of the experimental fish according to the dose of 10-30 mu g/g fish weight, then injecting colchicine solution according to the dose of 1-2 mu g/g fish weight, taking out the head and kidney after 1-2h, and placing the head and kidney in normal saline for later use;
(2) preparing a kidney cell suspension, cleaning the head and kidney tissue taken out in the step (1), removing blood clots and other tissues, then placing the head and kidney tissue in a cell screen, placing the cell screen in a culture dish containing KCl hypotonic solution, repeatedly shredding the head and kidney tissue placed in the cell screen, taking out the cell screen, blowing the hypotonic solution containing the head and kidney cells for 1-2 minutes, and standing to prepare the cell suspension;
(3) performing hypotonic treatment, namely taking culture dishes, placing glass slides in each culture dish, dropwise adding the cell suspension prepared in the step (2) onto the glass slides in the culture dishes, adding KCl hypotonic solution onto the glass slides, covering a culture dish cover, and performing hypotonic treatment for 30-40 minutes;
(4) fixing, namely after hypotonic treatment is finished, adding 5-6mL of freshly prepared Carnoy fixing liquid into the culture dish for fixing for 25-35 minutes, replacing the freshly prepared Carnoy fixing liquid, fixing for 1-2 hours, taking out the glass slide, flushing the glass slide for 2-3 times by using the freshly prepared Carnoy fixing liquid, and then placing the glass slide in the air for drying;
(5) and (3) dyeing, namely after the slide is completely dried, carrying out dyeing treatment for 5-10 minutes, then washing the slide clean with distilled water, and airing to obtain the fish chromosome karyotype.
2. The method for preparing a simple karyotype of fish according to claim 1, wherein: in the step (1), the plant hemagglutinin is injected into the basal part of the pectoral fin of the experimental fish by adopting the in vivo injection method of the plant hemagglutinin.
3. The method for preparing a simple karyotype of fish according to claim 1, wherein: in the step (1), the plant hemagglutinin may be injected for 1 time 15-20h before the injection of the plant hemagglutinin, and the colchicine solution may be injected at the same time or at an interval of 1-2h while the plant hemagglutinin is injected for the second time.
4. The method for preparing a simple karyotype of fish according to claim 1, wherein: in the step (1), the colchicine solution is injected 4-6h after the plant hemagglutinin is injected.
5. The method for preparing a simple karyotype of fish according to claim 1, wherein: in the step (2), the aperture of the cell screen is 100 μm.
6. The method for preparing a simple karyotype of fish according to claim 1, wherein: in the step (2) and the step (3), the concentration of KCl hypotonic solution is 0.75 g/L.
7. The method for preparing a simple karyotype of fish according to claim 1, wherein: in the step (3), the cell suspension is sucked up through a dropper, and is dripped onto glass slides placed in a culture dish from a height of 20-30 cm, and 3 drops are dripped on each glass slide.
8. The method for preparing a simple karyotype of fish according to claim 1, wherein: in the step (4), the Carnot fixing solution is added to the culture dish in an amount of submerging the glass slide in the culture dish.
9. The method for preparing a simple karyotype of fish according to claim 1, wherein: in the step (4), the volume ratio of methanol to glacial acetic acid in the Carnot's stationary liquid is 3: 1.
10. The method for preparing a simple karyotype of fish according to claim 1, wherein: in the step (5), the dyeing treatment is carried out by using a Giemsa dyeing solution with the volume ratio of 10%.
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| 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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