An incubation device for staining zebrafish embryos or larvae

Abstract

The application discloses an incubation device for zebrafish embryo or juvenile fish staining, relates to the technical field of biology, and is characterized in that a light-proof box body top drop port cover is provided with a light-shielding top cover, a light-shielding bottom cover is detachably connected with the bottom of the light-proof box body and blocks a liquid discharge port, a water filtering layer is arranged in the light-proof box body, a liquid replacement channel is arranged on the sidewall of the light-proof box body, the bottom of the liquid replacement channel is communicated with the bottom of the light-proof box body, the light-proof box body is provided with a first joint and a second joint, and the first joint and the second joint are used for connecting a liquid feeding device and a liquid discharging device respectively. The light-proof box body, the light-shielding top cover and the light-shielding bottom cover form a light-proof darkroom, the liquid replacement channel is used for replacing liquid, the first joint and the second joint realize the circulating flow of the culture liquid in the light-proof box body, liquid replacement or pipetting does not need to open the light-shielding top cover, light-sensitive reagents are effectively prevented from being invalid, the water filtering layer prevents embryos or juvenile fish from passing through and provides working liquid to flow through, the juvenile fish does not need to be transferred in the liquid replacement process, and the damage rate of the juvenile fish is effectively reduced and mechanical stimulation is reduced.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and in particular to an incubation device for staining zebrafish embryos or juveniles. Background Technology

[0002] Zebrafish (Danio rerio) is a model organism widely used in developmental biology, drug screening, and toxicology research. Current methods for whole-body immunofluorescence staining of zebrafish embryos / juveniles primarily utilize culture dishes, which have the following drawbacks: light leakage during pipetting or medium changes leads to the inactivation of photosensitizing reagents (e.g., photoquenching of fluorescent dyes); frequent medium changes require transferring juveniles, increasing the juvenile injury rate; and mechanical stimulation causes mislocalization of target proteins.

[0003] Therefore, how to effectively avoid the failure of photosensitive reagents, reduce the damage rate of juvenile fish, and reduce mechanical stimulation are problems that need to be solved by those skilled in the art. Utility Model Content

[0004] The purpose of this application is to provide an incubation device for staining zebrafish embryos or juveniles, which solves the problems of light leakage causing photosensitizing reagent failure during pipetting or changing the medium, increased juvenile damage rate due to frequent medium changes requiring the transfer of juveniles, and mislocalization of target proteins caused by mechanical stimulation.

[0005] To solve the above-mentioned technical problems, this application provides an incubation device for staining zebrafish embryos or juveniles, comprising: a light-proof box, a light-proof top cover, a water filter layer, a light-proof bottom cover, and a liquid exchange channel;

[0006] The top of the light-proof box is provided with an inlet, and the bottom of the light-proof box is provided with a drain outlet. The inlet is covered with a light-proof top cover, and the light-proof bottom cover is detachably connected to the bottom of the light-proof box and seals the drain outlet. The filter layer is located inside the light-proof box and is used to prevent embryos or juvenile fish from passing through and to allow working fluid to flow through. The fluid exchange channel is located on the side wall of the light-proof box, and the bottom of the fluid exchange channel is connected to the bottom of the light-proof box. The light-proof box is provided with a first connector and a second connector, which are used to connect the infusion device and the drain device, respectively.

[0007] In one optional embodiment, the filter layer includes a plurality of spaced-apart transparent filter channels. The two ends of the transparent filter channels are connected to the inner sidewall of the light-shielding box. The bottom surface of the transparent filter channel is parallel to the bottom surface of the light-shielding box. The first side surface of the transparent filter channel is perpendicular to the bottom surface of the light-shielding box. The angle between the second side surface of the transparent filter channel and the bottom surface of the transparent filter channel is an acute angle. A confinement area for confining juvenile fish samples is formed between the first side surface of one transparent filter channel and the second side surface of the other transparent filter channel in two adjacent transparent filter channels.

[0008] In one optional embodiment, the filter layer further includes a first connecting strip and a second connecting strip, with the first end of each transparent filter channel connected to the first connecting strip and the second end of each transparent filter channel connected to the second connecting strip. The inner sidewall of the light-proof box is provided with a first strip groove that engages with the first connecting strip and a second strip groove that engages with the second connecting strip.

[0009] In one optional embodiment, the first connecting strip is provided with a plurality of first slots, the plurality of first slots being spaced apart along the length direction of the first connecting strip; the second connecting strip is provided with a plurality of second slots, the plurality of second slots being spaced apart along the length direction of the second connecting strip; and the first end and the second end of the transparent filter channel are respectively inserted into the first slot and the second slot.

[0010] In one alternative embodiment, the first connecting strip and the second connecting strip are provided with connecting ears.

[0011] In one optional embodiment, the drain port has an external thread around its edge, and the light-shielding bottom cover has an internal thread that matches the external thread. The light-shielding bottom cover is detachably connected to the bottom of the light-shielding box via the external thread and the internal thread.

[0012] In one alternative embodiment, the edges of the light-shielding top cover and the inner threaded edges of the light-shielding bottom cover are provided with sealing rings.

[0013] In one optional embodiment, the light-shielding box is fitted with a heating jacket on its exterior, and a temperature sensor is provided inside the light-shielding box.

[0014] In one alternative embodiment, the first connector is located at the bottom of the light-shielding enclosure, and the second connector is located at the top of the light-shielding enclosure.

[0015] In one alternative embodiment, the first connector and the second connector are Luer connectors, the outer peripheral surface of which is coated with a light-shielding coating.

[0016] The present application provides an incubation device for staining zebrafish embryos or juveniles, comprising: a light-proof box, a light-proof top cover, a filter layer, a light-proof bottom cover, and a liquid exchange channel; the top of the light-proof box is provided with an inlet, the bottom of the light-proof box is provided with a drain outlet, the inlet cover is provided with a light-proof top cover, the light-proof bottom cover is detachably connected to the bottom of the light-proof box and seals the drain outlet, the filter layer is provided inside the light-proof box, the filter layer is used to prevent embryos or juveniles from passing through and to allow working fluid to flow through, the liquid exchange channel is located on the side wall of the light-proof box, the bottom of the liquid exchange channel is connected to the bottom of the light-proof box, and the light-proof box is provided with a first connector and a second connector, the first connector and the second connector being used to connect an infusion device and a drain device, respectively. The light-proof box, light-proof top cover, and light-proof bottom cover together form a light-proof dark chamber. The medium is changed through the medium exchange channel, and the culture medium in the light-proof box is circulated through the first and second connectors. The light-proof top cover does not need to be opened for medium change or transfer, which effectively avoids the failure of photosensitizing reagents. The filter layer prevents embryos or juvenile fish from passing through and allows working medium to flow through. There is no need to transfer juvenile fish during the medium change process, which can effectively reduce the damage rate of juvenile fish and reduce mechanical stimulation that leads to mislocalization of target proteins. Attached Figure Description

[0017] To more clearly illustrate the embodiments of this application, the accompanying drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 A structural diagram of an incubation device for staining zebrafish embryos or juveniles provided in an embodiment of this application;

[0019] Figure 2 This is a schematic diagram of a filter layer in an incubation device provided in an embodiment of this application.

[0020] The attached diagram is labeled as follows: 1-Light-proof box, 2-Light-proof top cover, 3-Light-proof bottom cover, 4-Liquid exchange channel, 5-First connector, 6-Second connector, 7-Transparent filter channel, 701-First side, 702-Second side. Detailed Implementation

[0021] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the protection scope of this application.

[0022] The core of this application is to provide an incubation device for staining zebrafish embryos or juveniles, which effectively avoids the failure of photosensitizing reagents, reduces the damage rate of juveniles, and reduces the mislocalization of target proteins caused by mechanical stimulation.

[0023] To enable those skilled in the art to better understand the present application, the present application will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0024] Figure 1 A structural diagram of an incubation device for staining zebrafish embryos or juveniles provided in this application embodiment is shown below. Figure 1 As shown, an incubation device for staining zebrafish embryos or juveniles includes: a light-proof box 1, a light-proof top cover 2, a filter layer, a light-proof bottom cover 3, and a liquid exchange channel 4; the top of the light-proof box 1 is provided with an inlet, and the bottom of the light-proof box 1 is provided with a drain outlet. The inlet is covered with a light-proof top cover 2. The light-proof bottom cover 3 is detachably connected to the bottom of the light-proof box 1 and seals the drain outlet. The filter layer is located inside the light-proof box 1. The filter layer is used to prevent embryos or juveniles from passing through and to allow working fluid to flow through. The liquid exchange channel 4 is located on the side wall of the light-proof box 1. The bottom of the liquid exchange channel 4 is connected to the bottom of the light-proof box 1. The light-proof box 1 is provided with a first connector 5 and a second connector 6. The first connector 5 and the second connector 6 are used to connect the infusion device and the drain device, respectively.

[0025] This application embodiment does not specifically limit the shape and structure of the light-shielding box 1. The material of the light-shielding box 1 can be black acrylonitrile butadiene styrene copolymer (ABS), which is a common thermoplastic engineering plastic. The top of the light-shielding box 1 has a filling port, and the bottom of the light-shielding box 1 has a drain port. A light-shielding top cover 2 is located at the filling port and can be opened and closed to seal the filling port and ensure a light-shielding environment. A light-shielding bottom cover 3 is used to block the drain port. The light-shielding bottom cover 3 is detachably connected to the light-shielding box 1, which can be achieved by a threaded connection. Specifically, the edge of the drain port has an external thread, and the light-shielding bottom cover 3 has an internal thread that matches the external thread. The light-shielding bottom cover 3 is detachably connected to the bottom of the light-shielding box 1 through the external and internal threads. Sealing rings are provided on the edges of the light-shielding top cover 2 and the internal thread edges of the light-shielding bottom cover 3 to improve the sealing effect of the light-shielding box 1. The light-shielding box 1, the light-shielding top cover 2, and the light-shielding bottom cover 3 together form a dark chamber to prevent the fluorescent dye from being quenched by light during the staining process. A filter layer is located inside the light-shielding box 1. The specific structure of the filter layer is not limited in this embodiment; it can have a microporous structure, allowing the working solution to flow freely while effectively blocking the passage of embryos or juvenile fish to prevent sample loss. The working solution can be the culture medium, fixative, or staining solution used in the incubation process. A liquid exchange channel 4 is located on the side wall of the light-shielding box 1. The bottom of the liquid exchange channel 4 is connected to the lowest point inside the light-shielding box 1, and the top of the liquid exchange channel 4 can be flush with the top of the light-shielding box 1. Liquid can be injected and drawn into the light-shielding box 1 through the liquid exchange channel 4, thus completing the liquid exchange without opening the light-shielding top cover 2. The liquid exchange channel 4 can also be made of a light-shielding material. The light-proof housing 1 is equipped with a first connector 5 and a second connector 6; alternatively, the first connector 5 can be connected to the infusion device and the second connector 6 can be connected to the drainage device, or the first connector 5 can be connected to the drainage device and the second connector 6 can be connected to the infusion device; the drainage device and the infusion device can be a pump or a syringe, etc.

[0026] The incubation device for staining zebrafish embryos or juveniles provided in this application includes: a light-proof box 1, a light-proof top cover 2, a filter layer, a light-proof bottom cover 3, and a liquid exchange channel 4; the top of the light-proof box 1 is provided with an inlet, and the bottom of the light-proof box 1 is provided with a drain outlet. The inlet is covered with a light-proof top cover 2. The light-proof bottom cover 3 is detachably connected to the bottom of the light-proof box 1 and blocks the drain outlet. The filter layer is located inside the light-proof box 1. The filter layer is used to prevent embryos or juveniles from passing through and to allow working fluid to flow through. The liquid exchange channel 4 is located on the side wall of the light-proof box 1. The bottom of the liquid exchange channel 4 is connected to the bottom of the light-proof box 1. The light-proof box 1 is provided with a first connector 5 and a second connector 6. The first connector 5 and the second connector 6 are used to connect an infusion device and a drain device, respectively. The light-proof box 1, the light-proof top cover 2, and the light-proof bottom cover 3 together form a light-proof dark chamber. The medium is changed through the medium change channel 4. The culture medium in the light-proof box 1 is circulated through the first connector 5 and the second connector 6. The light-proof top cover 2 does not need to be opened for medium change or transfer, which effectively avoids the failure of photosensitizing reagents. The filter layer prevents embryos or juvenile fish from passing through and allows the working medium to flow through. There is no need to transfer juvenile fish during the medium change process, which can effectively reduce the damage rate of juvenile fish and reduce mechanical stimulation that leads to mislocalization of target proteins.

[0027] Based on the above embodiments, Figure 2 This is a schematic diagram of a filter layer in an incubation device provided in an embodiment of this application, as shown below. Figure 1 and Figure 2 As shown, the filter layer includes multiple transparent filter channels 7 arranged at intervals. The two ends of the transparent filter channels 7 are connected to the inner sidewall of the light-shielding box 1. The bottom surface of the transparent filter channel 7 is parallel to the bottom surface of the light-shielding box 1. The first side surface 701 of the transparent filter channel 7 is perpendicular to the bottom surface of the light-shielding box 1. The angle between the second side surface 702 of the transparent filter channel 7 and the bottom surface of the transparent filter channel 7 is an acute angle. A confinement area for confining juvenile fish samples is formed between the first side surface 701 of one transparent filter channel 7 and the second side surface 702 of the other transparent filter channel 7.

[0028] In this embodiment, multiple transparent filter channels 7 are arranged at intervals, with the intervals used to allow the working fluid to flow through. The transparent filter channels 7 can be made of transparent acrylic material with high light transmittance. The bottom surface of the transparent filter channel 7 is parallel to the bottom surface of the light-shielding box 1, the first side surface 701 of the transparent filter channel 7 is perpendicular to the bottom surface of the light-shielding box 1, and the angle between the second side surface 702 of the transparent filter channel 7 and the bottom surface of the transparent filter channel 7 is an acute angle. Figure 1 As shown, the cross-section of the transparent filter channel 7 is a right-angled triangle. After the juvenile fish sinks, it automatically slides into the limiting area formed between the first side 701 of one transparent filter channel 7 and the second side 702 of the other transparent filter channel 7. The limiting area serves to physically lock the juvenile fish sample and also helps to ensure the stability of the sample during subsequent imaging.

[0029] Based on the above embodiments, the water filter layer in this application embodiment further includes a first connecting strip and a second connecting strip. The first end of each transparent water filter channel 7 is connected to the first connecting strip, and the second end of each transparent water filter channel 7 is connected to the second connecting strip. The inner side wall of the light-proof box 1 is provided with a first strip groove that engages with the first connecting strip and a second strip groove that engages with the second connecting strip.

[0030] This application embodiment does not limit the shape of the first and second connecting strips. The shapes of the first and second connecting strips depend on the shape of the light-shielding box 1. If the light-shielding box 1 is cylindrical, the first and second connecting strips are arc-shaped and fit against the inner wall of the light-shielding box 1. The transparent filter channel 7 is detachably connected to the light-shielding box 1 by engaging the connecting strip with the strip-shaped slot, allowing for the replacement of the filter layer according to the size of the individual sample. The spacing between the transparent filter channels 7 varies in different filter layers. Furthermore, the first and second connecting strips are provided with connecting ears, which can be used to engage with tweezers, allowing workers to use tweezers to grasp the connecting ears and remove the entire filter layer from the light-shielding box 1.

[0031] Based on the above embodiments, in this application embodiment, the first connecting strip is provided with a plurality of first slots, which are spaced apart along the length direction of the first connecting strip; the second connecting strip is provided with a plurality of second slots, which are spaced apart along the length direction of the second connecting strip; the first end and the second end of the transparent water filter channel 7 are respectively inserted into the first slot and the second slot. In this application embodiment, the two ends of the transparent water filter channel 7 are respectively inserted into the first connecting strip and the second connecting strip, which facilitates disassembly and subsequent cleaning.

[0032] Based on the above embodiments, the light-shielding box 1 in this application embodiment is externally fitted with a heating jacket, and a temperature sensor is provided inside the light-shielding box 1. The heating jacket may contain heating wires for heating the light-shielding box 1, and the temperature sensor is used to monitor the temperature inside the light-shielding box 1 so that the control system can adjust the power of the heating wires based on the temperature feedback from the temperature sensor, thereby achieving precise temperature control and maintaining a constant temperature environment inside the box.

[0033] Based on the above embodiments, in this application embodiment, the first connector 5 is located at the bottom of the light-shielding box 1, and the second connector 6 is located at the top of the light-shielding box 1. Of course, both the first connector 5 and the second connector 6 can also be located at the bottom of the light-shielding box 1. If the first connector 5 is located at the bottom of the light-shielding box 1, it is connected to an infusion device (pump). If the second connector 6 is located at the top of the light-shielding box 1, it is connected to a drainage device (drainage pipe and a storage tank connected to the drainage pipe). Liquid slowly enters from the bottom, pushing the existing liquid upwards to discharge, while waste or old liquid is discharged from the top, preventing liquid backflow or mixing. This low-in, high-out flow pattern simulates laminar flow, which is beneficial for maintaining the continuity and stability of liquid renewal. The slow, bottom-up flow pattern causes minimal disturbance to the juvenile fish samples, helping to keep them stably fixed on the filter layer. Furthermore, the first connector 5 and the second connector 6 are Luer connectors, with a light-shielding coating on their outer circumference. The Luer connectors are plug-and-play with laboratory equipment such as syringes, micropumps, and tubing, requiring no adapters. The outer circumference of the Luer connector is coated with a light-shielding coating (black glue or epoxy black paint) to eliminate the risk of light leakage at the connector, maintain a completely light-shielded environment inside the light-shielding box 1, and prevent the photosensitive reagent from becoming ineffective.

[0034] To better understand this application, the workflow of the incubation device based on zebrafish embryo or juvenile staining is described below.

[0035] Device assembly: Place the filter layer inside the light-proof box 1.

[0036] Embryo collection: The rearing water containing the embryos is poured in through the inlet; the embryos settle above the transparent filter channel 7 of the filter layer. Since the juvenile fish sample is larger than the interval between the transparent filter channels 7, the juvenile fish sample can be relatively stably fixed in the confinement area formed between the first side 701 of one transparent filter channel 7 and the second side 702 of the other transparent filter channel 7, which also helps to ensure the stability of the sample during subsequent imaging.

[0037] Live culture: Connect the light-shielding bottom cover 3 to the bottom of the light-shielding chamber 1; circulate the E3 culture medium through the first and second interfaces at a flow rate of 5 mL / h for 72 hours (the culture time can be adjusted according to experimental needs); close the light-shielding top cover 2 to form a sealed dark chamber. The E3 culture medium is the most commonly used standard culture medium for zebrafish embryos and juveniles, used to maintain their normal development and physiological state.

[0038] Fixation: After evacuating the culture medium through the medium exchange channel 4, inject 4% paraformaldehyde pre-cooled at 4°C; after standing for 30 minutes, inject phosphate-buffered saline (PBS) at a flow rate of 1 mL / min and wash 3 times (10 mL each time).

[0039] Staining in darkness: Inject 0.5% Triton X-100 through the media exchange channel 4 or the first interface and allow to stand for 10 min to enhance tissue permeability; dilute the primary antibody, such as Anti-GFP (green fluorescent protein antibody), at 1:500 and incubate at 37°C in the dark for 2 h; inject PBS at a flow rate of 1 mL / min and wash 3 times (10 mL each time); dilute the secondary antibody, such as Alexa Fluor 594 (fluorescent secondary antibody 594), at 1:1000 and incubate in the dark for 1 h; inject PBS at a flow rate of 1 mL / min and wash 3 times (10 mL each time); ensure that the liquid level evenly covers the embryo during each incubation.

[0040] Microscopic observation: Remove the bottom cover 3 and the top cover 2 to expose the filter layer. At this time, the sample is still stably fixed in the filter layer. Use tweezers to pick up the connecting ears at the edge of the filter layer and transfer them to the microscope stage. Use a high-power objective lens to observe them closely.

[0041] This application constructs a light-proof box 1 made of black engineering plastic, along with a light-proof top cover 2, a light-proof bottom cover 3, and a sealing ring, forming a physical light-proof system. Liquid transfer and replacement are completed through the liquid replacement channel 4, the first connector 5, and the second connector 6, without needing to open the light-proof top cover 2. This ensures that the entire process from cultivation, fixation, staining to cleaning is carried out in a light-proof environment, fundamentally eliminating the risk of light leakage and guaranteeing the stability of photosensitive reagents and the reproducibility of experimental results. This application also introduces a transparent acrylic filter layer, allowing juvenile fish to sink and naturally fix themselves between the inclined and vertical surfaces of the transparent filter channel 7 in the initial stage. Throughout the experiment, there is no need to move the sample; all reagent replacements are slowly injected or extracted through the first connector 5 and the second connector 6. The juvenile fish remain in place, avoiding any direct contact or mechanical disturbance. This "transfer-free" design significantly reduces the sample damage rate, while also reducing experimental errors caused by human operation, improving the accuracy and consistency of staining results. The transparent filter channel 7 of this application adopts a design with one side inclined and one side vertical, which guides the liquid to form a directional flow path, reduces eddies and stagnant areas, and enables the liquid to flow smoothly and evenly through the sample area, ensuring that residual reagents are thoroughly cleaned every time the liquid is changed, thus solving the problems of incomplete liquid change and poor flow in traditional devices.

[0042] The above provides a detailed description of an incubation device for staining zebrafish embryos or juveniles provided in this application. The various embodiments in the specification are described in a progressive manner, with each embodiment focusing on its differences from other embodiments. Similar or identical parts between embodiments can be referred to interchangeably. It should be noted that those skilled in the art can make various improvements and modifications to this application without departing from its principles, and these improvements and modifications also fall within the protection scope of the claims of this application.

[0043] It should also be noted that, in this specification, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

Claims

1. An incubation device for staining zebrafish embryos or juveniles, characterized in that, include: Light-proof box (1), light-proof top cover (2), water filter layer, light-proof bottom cover (3), and liquid exchange channel (4); The top of the light-proof box (1) is provided with an inlet, and the bottom of the light-proof box (1) is provided with a drain outlet. The inlet is covered with the light-proof top cover (2). The light-proof bottom cover (3) is detachably connected to the bottom of the light-proof box (1) and seals the drain outlet. The filter layer is located inside the light-proof box (1). The filter layer is used to prevent embryos or juvenile fish from passing through and to allow working fluid to flow through. The fluid exchange channel (4) is located on the side wall of the light-proof box (1). The bottom of the fluid exchange channel (4) is connected to the bottom of the light-proof box (1). The light-proof box (1) is provided with a first connector (5) and a second connector (6). The first connector (5) and the second connector (6) are used to connect the infusion device and the drain device, respectively.

2. The incubation device for staining zebrafish embryos or juveniles according to claim 1, characterized in that, The filter layer includes multiple transparent filter channels (7) arranged at intervals. The two ends of the transparent filter channels (7) are connected to the inner wall of the light-shielding box (1). The bottom surface of the transparent filter channel (7) is parallel to the bottom surface of the light-shielding box (1). The first side surface (701) of the transparent filter channel (7) is perpendicular to the bottom surface of the light-shielding box (1). The angle between the second side surface (702) of the transparent filter channel (7) and the bottom surface of the transparent filter channel (7) is an acute angle. A limiting area for limiting juvenile fish samples is formed between the first side surface (701) of one transparent filter channel (7) and the second side surface (702) of the other transparent filter channel (7).

3. The incubation device for staining zebrafish embryos or juveniles according to claim 2, characterized in that, The filter layer also includes a first connecting strip and a second connecting strip. The first end of each of the transparent filter channels (7) is connected to the first connecting strip, and the second end of each of the transparent filter channels (7) is connected to the second connecting strip. The inner side wall of the light-proof box (1) is provided with a first strip groove that engages with the first connecting strip and a second strip groove that engages with the second connecting strip.

4. The incubation device for staining zebrafish embryos or juveniles according to claim 3, characterized in that, The first connecting strip is provided with a plurality of first slots, which are spaced apart along the length of the first connecting strip. The second connecting strip is provided with a plurality of second slots, which are spaced apart along the length of the second connecting strip. The first end and the second end of the transparent filter channel (7) are respectively inserted into the first slot and the second slot.

5. The incubation device for staining zebrafish embryos or juveniles according to claim 4, characterized in that, The first and second connecting strips are provided with connecting ears.

6. The incubation device for staining zebrafish embryos or juveniles according to claim 1, characterized in that, The drain port has an external thread around its edge, and the light-shielding bottom cover (3) has an internal thread that matches the external thread. The light-shielding bottom cover (3) and the bottom of the light-shielding box (1) are detachably connected by the external thread and the internal thread.

7. The incubation apparatus for staining zebrafish embryos or juveniles according to claim 6, characterized in that, The edge of the light-shielding top cover (2) and the inner thread edge of the light-shielding bottom cover (3) are provided with sealing rings.

8. The incubation apparatus for staining zebrafish embryos or juveniles according to claim 5, characterized in that, The light-shielding box (1) is fitted with a heating jacket on the outside, and a temperature sensor is provided inside the light-shielding box (1).

9. The incubation device for staining zebrafish embryos or juveniles according to claim 1, characterized in that, The first connector (5) is located at the bottom of the light-shielding box (1), and the second connector (6) is located at the top of the light-shielding box (1).

10. The incubation apparatus for staining zebrafish embryos or juveniles according to claim 9, characterized in that, The first connector (5) and the second connector (6) are Luer connectors, and the outer peripheral surface of the Luer connector is coated with a light-shielding coating.

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