阅读说明：本技术 一种斑马鱼幼鱼固定装置及其固定方法 (Zebra fish juvenile fish fixing device and fixing method thereof ) 是由 卢曼慧 柴艳兵 张耀广 李兴佳 李宏 杨晓燕 于 2021-06-01 设计创作，主要内容包括：本发明公开了一种斑马鱼幼鱼固定装置及其固定方法,属于生物实验技术领域。包括孔板本体,孔板本体上设有凹槽,凹槽内均固设有用于固定斑马鱼的固定槽,凹槽的上表面高于固定槽的上表面,孔板本体和固定槽均为透明材质。本发明的固定方法为,在固定槽中加入鱼麻醉剂和黏着剂,用吸管吸取出生3～5天的斑马鱼幼鱼,蛋白酶去卵膜,预麻醉后放入固定槽中,用尖头镊将斑马鱼幼鱼拨至两眼重合的侧卧位。本发明的固定装置可快速整体移动样品进行观测,实现高通量快速检测,兼容性好,还可用于宽场成像,能将斑马鱼幼鱼摆放的方向一致,在观察时便于寻找斑马鱼,夹持的精准度高,不会损伤幼鱼鱼体,检测准确,适用于固定所有出生3～5天的斑马鱼幼鱼。(The invention discloses a zebra fish juvenile fish fixing device and a zebra fish juvenile fish fixing method, and belongs to the technical field of biological experiments. Including the orifice plate body, be equipped with the recess on the orifice plate body, all set firmly the fixed slot that is used for fixed zebra fish in the recess, the upper surface of recess is higher than the upper surface of fixed slot, and orifice plate body and fixed slot are transparent material. The fixing method comprises the steps of adding a fish anesthetic and an adhesive into a fixing groove, sucking the zebra fish juvenile fish born for 3-5 days by using a suction pipe, removing an egg membrane by using protease, placing the zebra fish juvenile fish into the fixing groove after pre-anesthesia, and pulling the zebra fish juvenile fish to a lateral position with two superposed eyes by using pointed forceps. The fixing device provided by the invention can be used for quickly moving the sample as a whole to observe, realizing high-flux quick detection, has good compatibility, can also be used for wide-field imaging, can be used for keeping the zebra fish juvenile fish in the same direction, is convenient to find the zebra fish during observation, has high clamping accuracy, can not damage the juvenile fish body, is accurate in detection, and is suitable for fixing all zebra fish juvenile fishes born for 3-5 days.)

1. The utility model provides a zebra fish juvenile fish fixing device, includes the orifice plate body, its characterized in that: the novel zebra fish anti-theft device is characterized in that a plurality of grooves are formed in the pore plate body, fixing grooves for fixing zebra fish are fixedly formed in each groove, the upper surfaces of the grooves are higher than the upper surfaces of the fixing grooves, and the pore plate body and the fixing grooves are made of transparent materials.

2. The zebrafish juvenile fish fixing device of claim 1, characterized in that: the groove is provided with eight groups of twelve grooves, each group of twelve grooves is cylindrical, the diameter of each groove is 6.5-7.0 mm, the depth of each groove is 5.5mm, and the transverse pitch and the longitudinal pitch are 8.9-9.1 mm.

3. The zebrafish juvenile fish fixing device of claim 2, characterized in that: the both ends of fixed slot are semi-circular, and tangent with the inner wall of recess, two curb plates of fixed slot are parallel to each other, the width of fixed slot is 6.3 ~ 6.7mm, and the degree of depth is 4.8 ~ 5.2mm, fixed tank bottom plate thickness is 0.45 ~ 0.55 mm.

4. A zebrafish juvenile fish fixing device according to any one of claims 1-3, characterized in that: the material of orifice plate body is transparent ya keli or transparent silica gel.

5. A method for fixing zebra fish juvenile fish is characterized by comprising the following steps: when the zebra fish juvenile fish is fixed, the method comprises the following steps:

the method comprises the following steps: selecting young zebra fish born for 3-5 days, adding protease, and removing egg membranes of the young zebra fish which is not hatched to form membranes;

step two: sucking the zebra fish juvenile fish by using a suction pipe, discharging redundant water in the suction pipe, and putting a fish anesthetic into the suction pipe for pre-anesthetizing for 10-20 s;

step three: taking the zebra fish juvenile fish fixing device of claims 1-4, adding a fish anesthetic and an adhesive into the fixing groove, and then putting the zebra fish juvenile fish after pre-anesthesia;

step four: and (4) moving the zebra fish juvenile fish to a lateral position with two superposed eyes by using a pointed forceps, and carrying out subsequent observation after the adhesive is solidified.

6. The method for fixing the zebra fish juvenile fish according to claim 5, wherein: the preparation method of the adhesive comprises the following steps: adding 0.2-0.3 part of agar into 100 parts of fish anesthetic solution by mass, and heating to 90-100 ℃ to form 0.2-0.3% solution; when the zebra fish juvenile fish is placed in the fixing groove, the temperature of the fish anesthetic and the agar is kept between 28 ℃ and 32 ℃.

7. The method for fixing the zebra fish juvenile fish according to claim 5, wherein: adding 0.2-0.3 part of agarose into 100 parts of fish anesthetic solution by mass, and heating to 90-100 ℃ to form 0.2-0.3% solution; when the zebra fish juvenile fish is placed in a fixed groove, the temperature of the fish anesthetic and the temperature of the agarose are kept between 28 ℃ and 32 ℃.

8. The method for fixing the zebra fish juvenile fish according to claim 5, wherein: the preparation method of the adhesive comprises the following steps: adding 2-3 parts of methylcellulose into 100 parts of fish anesthetic solution by mass to form a solution with the concentration of 2-3%.

Technical Field

The invention belongs to the technical field of biological experiments, relates to a zebra fish juvenile fish fixing device and further relates to a zebra fish juvenile fish fixing method.

Background

The zebra fish is a small spine model organism, the homology of the gene and the human genome is up to 87%, the zebra fish has unique biological characteristics and genetic advantages, has the advantages of simple feeding, short breeding period, transparent embryo, large egg laying amount, easiness in collection and the like, and is widely applied to many fields.

Since the whole zebra fish juvenile fish is transparent, many studies directly utilize the zebra fish juvenile fish for in vivo observation. In the experimental process, an experimenter generally narcotizes the zebra fish juvenile fish by using an anesthetic, then transfers the zebra fish juvenile fish into a methyl cellulose solution by using a suction pipe, and then swings the zebra fish juvenile fish body position by using a needle or a pair of tweezers until two eyes coincide, and finally, observation and shooting are carried out. The method for fixing the zebra fish juvenile fish has the following defects: 1. the zebra fish juvenile fish is small in size and generally only about 2-3mm, and the zebra fish juvenile fish moves at a high speed and can move in methyl cellulose with high resistance, so that the position of the fish body needs to be repeatedly placed, the workload of experimenters is increased, the juvenile fish body can be damaged, whether a malformed structure is caused by a compound or not is difficult to judge, and the accuracy of a result is influenced; 2. the number of the zebra fish juvenile fishes in each group of experiment is large, and the time and the labor are consumed for fixing the fish body in the experiment process.

Therefore, a simple and rapid zebra fish juvenile fish fixing device and a fixing method thereof are urgently needed to be designed, so that the experiment efficiency is improved, and the experiment accuracy is ensured.

Disclosure of Invention

In order to solve the defects in the prior art, the invention aims to provide a zebra fish juvenile fish fixing device so as to achieve the purposes of quickly moving a sample integrally for observation and quickly detecting at high flux;

the invention also provides a zebra fish juvenile fish fixing method to achieve the purposes of not damaging juvenile fish, improving detection accuracy and quickly fixing zebra fish juvenile fish in the fixing process.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows: the utility model provides a zebra fish juvenile fish fixing device, includes the orifice plate body, be equipped with a plurality of recesses on the orifice plate body, all set firmly the fixed slot that is used for fixed zebra fish in every recess, the upper surface of recess is higher than the upper surface of fixed slot, orifice plate body and fixed slot are transparent material.

As a limitation of the present invention: the groove is provided with eight groups of twelve grooves, each group of twelve grooves is cylindrical, the diameter of each groove is 6.5-7.0 mm, the depth of each groove is 5.5mm, and the transverse pitch and the longitudinal pitch are 8.9-9.1 mm.

As a further limitation of the invention: the both ends of fixed slot are semi-circular, and tangent with the inner wall of recess, two curb plates of fixed slot are parallel to each other, the width of fixed slot is 6.3 ~ 6.7mm, and the degree of depth is 4.8 ~ 5.2mm, fixed tank bottom plate thickness is 0.45 ~ 0.55 mm.

As another limitation of the present invention: the material of orifice plate body is transparent ya keli or transparent silica gel.

A zebra fish juvenile fish fixing method comprises the following steps:

the method comprises the following steps: selecting young zebra fish born for 3-5 days, adding protease, and removing egg membranes of the young zebra fish which is not hatched to form membranes;

step two: sucking the zebra fish juvenile fish by using a suction pipe, discharging redundant water in the suction pipe, and putting a fish anesthetic into the suction pipe for pre-anesthetizing for 10-20 s;

step three: taking the zebra fish juvenile fish fixing device of claims 1-4, adding a fish anesthetic and an adhesive into the fixing groove, and then putting the zebra fish juvenile fish after pre-anesthesia;

step four: and (4) moving the zebra fish juvenile fish to a lateral position with two superposed eyes by using a pointed forceps, and carrying out subsequent observation after the adhesive is solidified.

As a limitation of the adhesive of the present invention: the preparation method of the adhesive comprises the following steps: adding 0.2-0.3 part of agar into 100 parts of fish anesthetic solution by mass, and heating to 90-100 ℃ to form 0.2-0.3% solution; when the zebra fish juvenile fish is placed in the fixing groove, the temperature of the fish anesthetic and the agar is kept between 28 ℃ and 32 ℃.

As another limitation on the adhesive of the present invention: adding 0.2-0.3 part of agarose into 100 parts of fish anesthetic solution by mass, and heating to 90-100 ℃ to form 0.2-0.3% solution; when the zebra fish juvenile fish is placed in a fixed groove, the temperature of the fish anesthetic and the temperature of the agarose are kept between 28 ℃ and 32 ℃.

As a third limitation on the adhesive of the present invention: the preparation method of the adhesive comprises the following steps: adding 2-3 parts of methylcellulose into 100 parts of fish anesthetic solution by mass to form a solution with the concentration of 2-3%.

Due to the adoption of the technical scheme, compared with the prior art, the invention has the following beneficial effects:

(1) the fixing device can quickly and directionally place the zebra fish juvenile fish, the hole plate body is provided with the plurality of fixing grooves, so that the zebra fish juvenile fish can be fixed in a multi-person cooperation mode, the plurality of fixing grooves are formed in the hole plate body, a sample can be quickly and integrally moved for observation, high-flux quick detection can be realized, the compatibility is good, the fixing device can also be used for wide-field imaging, the fixing device is used in cooperation with most equipment, the universality is strong, the upper surfaces of the fixing grooves are lower than that of the grooves, when an adhesive in the fixing grooves overflows, the fixing device can be contained in the grooves, the adhesive is prevented from scattering on the surface of the hole plate body, and the transparent material is convenient to observe;

(2) the diameter and the pitch of the grooves are set to be specified sizes, the grooves are convenient to adapt to an observation instrument, the observation instrument can completely clamp ninety-six holes at one time, the observation efficiency is improved, the fixing groove is in a slender shape, when the zebra fish juvenile fish is fixed, the zebra fish juvenile fish is fixed along the length direction of the fixing groove according to requirements, the fixing direction of the zebra fish juvenile fish is ensured to be consistent, the observation is convenient, and the time for searching the zebra fish can be saved in the observation and shooting processes;

(3) the zebra fish is placed in the lateral position with two coincident eyes, static observation of organ development conditions in the zebra fish body and dynamic observation of heartbeat are realized for a long time through adhesive solidification and anesthesia of fish anesthetic, and as juvenile fish is anesthetized, clamping accuracy is high during fixing, juvenile fish bodies cannot be damaged, and detection accuracy is improved.

In conclusion, the fixing device can quickly and integrally move a sample to observe, high-flux quick detection is realized, the compatibility is good, the fixing device can also be used for wide-field imaging, when zebra fish juvenile fishes are fixed, the zebra fish juvenile fishes can be placed in the same direction, the zebra fish juvenile fishes can be conveniently found during observation, the zebra fish juvenile fishes can be completely fixed, the clamping accuracy is high, juvenile fish bodies cannot be damaged, the detection is accurate, and the fixing device is suitable for fixing all zebra fish juvenile fishes born for 3-5 days.

Drawings

The invention is described in further detail below with reference to the figures and the embodiments.

Fig. 1 is a schematic top view of the structure of embodiment 1 of the present invention;

FIG. 2 is a schematic cross-sectional view of the structure of FIG. 1A-A;

FIG. 3 is a schematic cross-sectional view of the structure of FIG. 1B-B.

In the figure: 1-orifice plate body, 2-groove, 3-fixed groove, 4-side plate, 5-bottom plate.

Detailed Description

Preferred embodiments of the present invention will be described below with reference to the accompanying drawings. It should be understood that the description of the zebra fish fry fixing device and the fixing method thereof is a preferred embodiment, which is only used for illustrating and explaining the present invention and is not to be construed as limiting the present invention.

Embodiment 1 zebra fish juvenile fish fixing device

This embodiment is as shown in fig. 1-fig. 3, a zebra fish juvenile fish fixing device, including orifice plate body 1, be equipped with a plurality of recesses 2 on the orifice plate body 1, orifice plate body 1 is transparent ya keli material, also can be transparent silica gel material, orifice plate body 1 is the cuboid, and its thickness is 9.0mm, recess 2 is inside sunken cylindrical, recess 2's diameter is 6.5mm, and inside sunken degree of depth is 5.5 mm. Eight groups of grooves 2 are arranged on the orifice plate body 1, each group is twelve, namely twelve grooves in the length direction and eight rows in the width direction, and the total number is ninety-six. The pitch between two adjacent grooves 2 is 9.0mm, namely the pitch between the grooves 2 adjacent in the transverse direction is 9.0mm, and the pitch between the grooves 2 adjacent in the longitudinal direction is 9.0 mm.

All set firmly fixed slot 3 that is used for fixed zebra fish in every recess 2, the upper surface of recess 2 is higher than the upper surface of fixed slot 3, and whole fixed slot 3 locates the inside of recess 2 promptly and does not expose. The both ends of fixed slot 3 are semi-circular, and tangent with the inner wall of recess 2, and the semi-circular outer wall at fixed slot 3 both ends is tangent with the inner wall of recess 2 promptly, fixes in recess 2 through the bonding, two curb plates 4 of fixed slot 3 are parallel to each other, the distance between two parallel curb plates 4 of fixed slot 3 is 6.5mm, and the width is 6.5mm promptly, and its degree of depth is 5.0mm, the thickness of 3 bottom plates 5 of fixed slot is 0.48 mm. The material of fixed slot 3 is transparent ya keli, also can be transparent silica gel.

Embodiment 2 zebra fish juvenile fish fixing device

The structure of this embodiment is the same as that of embodiment 2, except that the dimensional parameters of each part are set, and the specific dimensions are shown in table 1:

table 1 table of dimensional parameters of parts

Example 5 method for fixing zebra fish juvenile fish

A method for fixing zebra fish juvenile fishes comprises the steps of preparing any one of zebra fish juvenile fishes in embodiment 1-4, utilizing the zebra fish juvenile fish fixing device in embodiment 1, selecting zebra fish juvenile fishes born for 3-5 days when the zebra fish juvenile fishes are fixed, adding protease, removing egg membranes of the zebra fish juvenile fishes without membranes incubated, observing the zebra fish juvenile fishes, sucking the zebra fish juvenile fishes by using a suction pipe after the zebra fish juvenile fishes without membranes incubated are incubated out of the membranes, discharging excessive water in the suction pipe, putting a fish anesthetic into the suction pipe, and pre-anesthetizing for 14 s.

Adding a fish anesthetic and an adhesive into the fixing groove 3, then placing the preanesthesized zebra fish juvenile fish, moving the zebra fish juvenile fish to a lateral position with two eyes overlapped by using a pointed forceps, and carrying out subsequent observation after the adhesive is solidified.

The preparation method of the adhesive comprises the following steps: 2g of agar is added into 1000g of fish anesthetic solution, the agar is solid under the normal state, so that the agar needs to be heated to 90 ℃ and becomes liquid, and then the liquid is mixed with the fish anesthetic to form a solution with the concentration of 0.2 percent, when the zebra fish juvenile fish is placed into the fixing groove 3, the temperature of the fish anesthetic and the agar is kept between 30 ℃, the zebra fish juvenile fish cannot be damaged, the state of the agar can also play a role of resistance, and the zebra fish juvenile fish can be conveniently fixed.

Embodiment 6 zebra fish juvenile fish fixing method

This example is the same as the fixing method of example 5, and the time for pre-anaesthetizing the zebra fish juvenile fish in the straw and the preparation method of the adhesive are different, the time for pre-anaesthetizing the zebra fish juvenile fish in the straw is 15s, the adhesive of this example is made of agarose, and the preparation method is as follows: 2.5g of agarose is added into 1000g of fish anesthetic solution, the agarose is solid in a normal state, so the agarose needs to be heated to 97 ℃, the agarose becomes liquid and then is mixed with the fish anesthetic to form a solution with the concentration of 0.25%, when the zebra fish juvenile fish is placed into the fixing groove 3, the temperature of the fish anesthetic and the agarose is kept between 31 ℃, the zebra fish juvenile fish cannot be damaged, the state of the agarose can also play a role of resistance, and the zebra fish juvenile fish can be conveniently fixed.

Example 7 method for fixing zebra fish juvenile fish

This example is the same as the fixing method of example 5, and the time for pre-anaesthetizing the zebra fish juvenile fish in the straw and the preparation method of the adhesive are different, the time for pre-anaesthetizing the zebra fish juvenile fish in the straw is 16s, the adhesive of this example uses methyl cellulose, and the preparation method is as follows: 20g of methylcellulose were added to 1000g of the fish anesthetic solution to form a 2% strength solution.

Example 8-15 method for fixing zebra fish larvae

The example is the same as the example 5, except for setting various parameters, as shown in table 2:

TABLE 2

Example 16 demonstration of the injury to juvenile Zebra fish using the fixation method of example 5

Randomly selecting 192 zebra fish juvenile fishes born for 3-5 days, wherein 96 zebra fish juvenile fishes are placed in the zebra fish juvenile fish fixing device in the embodiment 1, one zebra fish juvenile fish fixing device is placed in each fixing groove 3, and then fixing the zebra fish juvenile fishes by using the fixing method in the embodiment 5; the remaining 96 zebra fish juvenile fishes are placed in a common fixing device, the zebra fish juvenile fishes are fixed by a common fixing method, in the fixing process, the damage conditions of workers on the zebra fish juvenile fishes during fixing are recorded, and after statistics, the records are recorded in table 2:

TABLE 2 injury during immobilization of zebra fish larvae

Categories	Number of injured zebra fish larvae (only)	Damage ratio (%)
			Zebrafish juvenile fish were fixed using the fixing method of example 5	2	0.02
Fixing zebra fish juvenile fish by using common fixing method	11	0.11

As can be seen from the table 2, the damage rate is small by utilizing the zebra fish juvenile fish fixed in the application, and the subsequent detection is more accurate.