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Jul 2020
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Brain-localized and Intravenous Microinjections in the Larval Zebrafish to Assess Innate Immune Response
对斑马鱼幼体进行脑局部和静脉显微注射以评估先天免疫反应   

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Abstract

Creating a robust and controlled infection model is imperative for studying the innate immune response. Leveraging the particular strengths of the zebrafish model system, such as optical transparency, ex utero development, and large clutch size, allows for the development of methods that yield consistent and reproducible results. We created a robust model for activation of innate immunity by microinjecting bacterial particles or live bacteria into larval zebrafish, unlike previous studies which largely restricted such manipulations to embryonic stages of zebrafish. The ability to introduce stimuli locally or systemically at larval stages provides significant advantages to examine host response in more mature tissues as well as the possibility to interrogate adaptive immunity at older larval stages. This protocol describes two distinct modes of microinjection to introduce lipopolysaccharide (LPS) or bacteria into the living larval zebrafish: one localized to the brain, and another into the bloodstream via the caudal vein plexus.


Graphic abstract:


Schematic shows the two distinct modes of larval zebrafish microinjection, either in the brain parenchyma or in the blood stream intravenously. Reagents introduced into the zebrafish to assess immune response are depicted in the “injection components” as described in the protocol.


Keywords: Macrophage (巨噬细胞), Microinjection (显微注射), Zebrafish (斑马鱼), Inflammation (炎症), Infection (感染), Immunology (免疫学), Brain (大脑), Circulation (循环)

Background

The complex interactions during an infection require the use of in vivo animal models to fully understand the dynamic interplay between the pathogen and its host. Studying this phenomenon requires a controlled and reliable method of pathogen delivery. Zebrafish has been used as a model for studying the immune response to a variety of pathogens (Menudier et al., 1996; Davis et al., 2002; Neely et al., 2002; Prouty et al., 2003; van der Sar et al., 2003; O'Toole et al., 2004; Phelan et al., 2005; Phelps and Neely, 2005; Pressley et al., 2005) for its evolutionarily conserved innate immune system (Herbomel et al., 1999; Traver et al., 2003; Trede et al., 2004), optical transparency, large embryo clutch sizes, genetic tractability and in vivo imaging capabilities (Kimmel, 1989; Kimmel et al., 1988 and 1995; Sullivan and Kim, 2008; Kanther and Rawls, 2010). Previous infection protocols have immersed fish in pathogens by directly adding it to fish water (Davis et al., 2002; O'Toole et al., 2004; Prouty et al., 2003) or injected bacteria into the axial vein or hindbrain ventricle in the early embryo at 28 h post- fertilization (hpf) (van der Sar et al., 2003), or injected LPS at later stages in the larval zebrafish at 3-6 days post-fertilization (dpf) into the yolk to induce a lethal systemic immune response (Yang et al., 2014). Extending from previously published methods, we developed a protocol for microinjection of LPS or bacteria in the larval zebrafish, either directly into the brain parenchyma or into blood circulation to cause a robust innate immune response starting in the 4 dpf larvae. Since adaptive immunity does not begin until about 4 weeks after fertilization in zebrafish (Davis et al., 2002), we can leverage the early larval stages of zebrafish to study specific innate immune functions independent of adaptive immunity. Co-injection of immune activators with a fluorescently labeled dextran, or direct injection of fluorescently tagged immune activators allows for a quick visual verification of a successful injection as well as subsequent labeling of the macrophage response based on phagocytosis of the reporter. This protocol describes two different modes of microinjection with distinct target sites: first, the caudal vein plexus for systemic distribution throughout blood flow, and second, the brain tectum that briefly localizes the injected substance in the brain but is subsequently drained out into circulation (Yang et al., 2020). Although we describe our protocol for 3-5 dpf larvae, these methods are applicable to later larval stages at least up to 10 dpf (Yang et al., 2020).


Materials and Reagents

  1. Polystyrene Petri dish (VWR, catalog number: 25384-342)

  2. 7.5 ml transfer pipettes (VWR, catalog number: 414004-005)

  3. Thin wall glass capillaries 4 with filament, OD 1.5 mm (World Precision Instruments, catalog number: TW150F-4)

  4. Escherichia coli (any strain)

  5. Lipopolysaccharides from Escherichia coli O111:B4 (Sigma-Aldrich, catalog number: L3024) (store at -20°C)

  6. Lipopolysaccharides from Escherichia coli Serotype 055:B5, Alexa FluorTM 594 Conjugate (Thermo Fisher Scientific, Invitrogen, catalog number: L23353) (store at -80 °C and protect from light)

  7. Dextran, Alexa FluorTM 568 (Thermo Fisher Scientific, Invitrogen, catalog number: D-22912) (store at -20 °C and protect from light)

  8. Phosphate-buffered saline (PBS), pH 7.4

  9. 1.5% low melt agarose (Fisher Scientific, IBI Scientific, catalog number: 50-550-455) (see Recipes)

  10. Tricaine (3-amino benzoic acid ethyl ester) (Sigma-Aldrich, catalog number: A-5040) (store at room temperature)

    Note: Store at -20 °C when made into 25× Tricaine solution (see Recipes).

  11. PTU (N-Phenylthiourea) (Sigma-Aldrich, catalog number: P7629) (store at room temperature)

    Note: Store at -20 °C when made into 50× PTU solution (see Recipes).

Equipment

  1. Programmable Horizontal Pipette Puller (World Precision Instruments, catalog number: PUL-1000)

  2. Pneumatic PicoPump (World Precision Instruments, catalog number: SYS-PV820)

  3. Fluorescent stereomicroscope fully apochromatic corrected with 16:5:1 zoom optics (Leica, model number: M165 MC)

  4. Incubator (Benchmark Scientific, model number: H2200-H)

  5. Stage Micrometer, 1 × 0.01 mm (AmScope, catalog number: MR095)

  6. Magnetic stand (World Precision Instruments, catalog number: M1)

  7. Manual micromanipulator (World Precision Instruments, catalog number: M3301)

  8. Steel base plate, 10 lbs (World Precision Instruments, catalog number: 5052)

  9. PicoNozzle Kit v2 (World Precision Instruments, catalog number: 5430-ALL)

  10. Dumont #55 Forceps (Fine Scientific Tools, catalog number: 11295-51)

Procedure

  1. Zebrafish embryo and larval husbandry

    1. Maintain zebrafish in Petri dishes with autoclaved fish water supplemented with 0.003% PTU and incubate at 28.5 °C.

      For general questions, the composition of fish water and regular zebrafish husbandry are well described elsewhere including Avdesh et al. (2012).

    2. Replace water with fresh water containing 0.003% PTU daily and monitor health.

    3. Begin experiment when embryos reach your desired larval stage (such as 4 dpf).


  2. Injection mixture preparations

    1. E. coli supplemented with fluorescent dextran: Prepare 3 ml overnight culture derived from a single colony. Centrifuge culture at 3,000 rpm for 1 min, remove supernatant and re-suspend in 500 μl of 1× PBS (pH 7.4). This should be approximately 1.6 × 106 cfu/μl. Add 1 μl of 5 ng/nl fluorescent dextran. Flick the tube with your finger and spin down before use.

    2. LPS supplemented with fluorescent dextran: Mix 9 μl of LPS at 5 ng/nl from Escherichia coli O111:B4 and 1 μl of a 1:10 dilution of 5 ng/nl fluorescently labeled dextran to make a final dilution of 1:100 supplementation with fluorescent dextran. Flick the tube with your finger to mix and spin down before use.

    3. LPS directly conjugated to fluorescent molecules (e.g., Alexa 594) – use directly at 5 ng/nl.

    4. Control vehicle injection using ultra-pure or autoclaved water supplemented with fluorescent dextran – mix 9 μl of water with 1 μl of a 1:10 dilution of 5 ng/nl fluorescently labeled dextran. Flick the tube with your finger to mix and spin down before use.


  3. Needle and microinjector set-up

    1. Microinjection needles are pulled from glass capillaries using a Programmable Horizontal Pipette Puller or similar equipment (Table 1).


      Table 1. 4-step protocol for glass capillary tube pulling
      Step Heat Force Distance Delay
      1 690 260 7.3 0
      2 500 240 0.5 4
      3 500 230 0.5 10
      4 380 240 0.5 20


    2. Load ~3 μl of injection solution into needle.

      Note: Unless a very fine pipette tip is used, the solution will remain at the mouth of the needle. To force the solution to the tip of the needle, hold needle firmly between middle finger and thumb and flick wrist in a downward motion (see Video 1). Repeat until liquid has reached the narrow tip of the needle.

      Video 1. Loading of injection mixture into a microinjection needle


    3. Transfer needle into the micromanipulator and set out of the way.


  4. Mounting zebrafish for microinjections (see Video 2)

    Video 2. Stepwise demonstration of mounting larvae for microinjection

    1. Use a plastic transfer pipette to transport the larvae to the center of a clean Petri dish lid and remove as much water as possible.

      Note: Petri dish lids are used because the sides of the lid have a low profile which allows more latitude to position the needle to the desired target. Several larvae can be mounted at once; for brain injections we can mount upwards of 20-30 zebrafish larvae at once.

    2. Heat 1.5% low-melt agarose (solid form) in microwave to melt it to a liquid form.

      Note: Approximately 20 s to less than 1 min is needed to melt 100 ml of agarose. Low melt agarose heats up very fast so you will want to stand nearby to monitor the heating to prevent boil over.

    3. Use pipette to collect a small amount of low-melt agarose.

      1. Immediately after microwaving, the agarose will be very hot. If you see steam/condensation within the pipette then the agarose is too hot and will burn the larvae. Periodically monitor the temperature of the agarose by carefully touching the outside of the pipette where agarose is located. The agarose should be warm to touch, not hot, and remain fluid.

        The low-melt agarose will remain fluid at 37 °C and set rapidly below 25 °C. Aim to use the agarose at a temperature between 30 °C and 40 °C. Keeping the agarose on a heat block at 60 °C will preserve the solution in a fluid state for a longer period of time as you work on mounting the larvae.

      2. Cooling the agarose can be expedited by pipetting the hot agarose onto a clean surface (such as another Petri dish) up and down a few times (see Video 2).

    4. Form a circle around the larvae to be injected with the agarose and go in smaller concentric circular motions until the agarose touches the larvae. Mix the agarose with the larvae by using the pipette to gently swirl them together or by pipetting them up and down 1-2 times (see Video 2).

      Note: For ease of injection, the goal is to embed the larvae in a thin layer of agarose which allows the fine glass needle to easily penetrate through the gel. Using too much agarose will make orienting the needle more difficult and cause unwanted bending of your glass needle.

    5. Use forceps to orient the larvae.

      1. This is the most time sensitive step because larvae must be correctly positioned before the agarose re-solidifies (1-2 min). Use fine forceps to quickly orient each larva, depending on desired injection site, but without concern for the exact orientation or body alignment. Be cautious to not injure the larvae by poking them, instead use the agarose around them to nudge and move them into position (see Video 2).

      2. For brain and intravenous injections, position the larvae on their dorsal or ventral-lateral sides, respectively.

    6. Wait for agarose to cool down and solidify before starting injections.

    7. Add a small amount of fish system water on top of the agarose to keep larvae in water and healthy during the operation.


  5. Setting up the pneumatic microinjector and calibrating your needle for injection

    1. Review the manufacturer’s recommendations and instructions for the micro-needle puller and the microinjector before operating.

    2. Turn on PicoPump and open air valve. The output pressure should be set to 20 psi. Check that you can feel air eject out from the needle holder (PicoNozzle Kit v2) before putting the needle in place.

    3. Insert your pulled glass needle. Prepare extra needles in case they break or when you need to re-calibrate.

    4. Looking through the microscope orient the tip of the needle into the middle of the field of view by adjusting the manual micromanipulator set on the magnetic stand on a steel base plate that is holding your needle.

    5. Use fine forceps to gently break off small controlled amounts of the glass needle. The objective is to break the minimum amount of the needle to allow the smallest amount of liquid to eject out (see Figure 1).



      Figure 1. Calibration of injection needle using a micrometer. To ensure a consist injection volume, use a micrometer to measure the diameter of the injection bubble volume. An injection bubble diameter of 100 μm is equivalent to 0.5 nl.


    6. Press the PicoPump foot pedal to see if a noticeable amount of liquid is visible on the tip of the needle. If liquid is not present, continue to carefully clip small pieces off the needle until you can see a tiny amount of liquid.

    7. Use a micrometer to calibrate the size of your needle opening (see Figure 1).

      1. Adjust the opening size of your glass needle by chipping off the end incrementally under the stereoscope while you test how much volume is ejected after each kick by estimating the spherical drop size at the needle tip in air right above a micrometer.

      2. Aim for the spherical drop to have a diameter of about 100 μm on the micrometer to give a 0.5 nl volume per kick on the PicoPump foot pedal (see Figure 1).

        Note: To prevent complications from the injection site minimize the size of the needle. We suggest using a smaller needle and administering a 1 nl injection with 2 kicks.

      3. The volume (V) of your droplet from your needle can be determined by the standard formula for a sphere volume (), where r is the radius of your droplet.


  6. Brain microinjection

    1. Conduct the microinjections under a fluorescent stereomicroscope in order to monitor and validate each successful injection by fluorescence. The mounted larvae in low-melt agarose will need to be oriented with brain side up for needle access (see Figure 2).

    2. Place needle directly above the brain tectum (see Figure 2).

    3. Use the micromanipulator to slowly puncture the skin with the needle.

      Note: Force of the puncture can drive the needle further into the brain than desired. Pull the needle backwards until the needle is only superficially piercing the brain.

    4. Press the PicoPump foot pedal twice to eject 1 nl.

    5. Screen for successful injection by visualizing fluorescence in the brain tectum at the site of injection (see Figure 2B).

      Note: It is normal for the injection liquid to spread into the hindbrain ventricle. Remove any larvae that are not injected correctly by using forceps to grab the larvae out of the agarose.



      Figure 2. Brain and intravenous microinjection sites. A. Red arrow points to brain injection site in the right tectum and white box outlines the right tectum region of the larval brain at 3 dpf. B. 30 s after fluorescent dextran injection into the brain tectum (red arrow), the fluorescent tracer can be readily observed to disperse into the brain ventricles and into the spinal canal (red arrowheads). C. Red arrow points to the intravenous injection site at the caudal vein plexus located beneath the dorsal aorta (top red arrowhead) and near the urogenital opening (bottom red arrowhead). D. Full body image 30 s after intravenous injection shows fluorescent dextran signal throughout the entire body vasculature. Red arrowheads point to vasculature in the head and trunk made visible by fluorescent dextran. White dotted box, region shown in E. E. Immediately following intravenous injection, the heart (red circle) is filled with fluorescent dextran signal. Red arrowheads point to fluorescent signal in the gill vasculature.


  7. Intravenous microinjection

    1. Conduct the microinjection under a fluorescent stereoscope in order to monitor and validate each successful injection by fluorescence (see Figure 2).

    2. Your mounted larvae in low-melt agarose will need to be oriented with ventral-lateral side up to access the yolk extension side of the tail to inject into the caudal vein plexus (see Figure 2C).

      Note: It is most effective to be able to visualize and aim needle into the thin strip of tissue at the caudal vein plexus, which is the most ventral side of the body wall immediately posterior to the end of the yolk extension where continuous circulation can be readily observed in the caudal vein (see Figure 2C).

    3. Place needle directly above the caudal vein plexus close to the urogenital opening (see Figure 2C).

    4. Use the micromanipulator to slowly puncture the skin with the needle.

      Note: The force of the puncture can drive the needle pass the vein and into the muscle. Pull the needle backwards until it is in the vein.

    5. Press the PicoPump foot pedal twice to eject 1 nl.

    6. After a successful injection, the immediate detection of fluorescence will happen in the heart (Figure 2E) and from there the injected substance will be widely circulated throughout the body vasculature which is entirely visible within seconds to 1 min after your injection (see Figure 2D-E).

      Note: Remove any larvae that are not injected correctly. Intravenous injection requires more technical practice than a brain injection since the target tissue (caudal vein plexus) is far thinner and smaller in area than the tectum.


  8. Post-injection larvae recovery

    1. Recover injected embryos by removing them from the agarose.

      1. Use the side of the forceps to create a break in the agarose beginning underneath the head.

      2. Drag the forceps along the body until the tip of the tail is reached.

      3. Larvae should be able to squirm out easily and swim into the fish water above the gel.

    2. Use transfer pipette to place injected larvae into a clean Petri dish with fresh fish water supplemented with PTU.

    3. Place larvae into 28.5 °C incubator to recover.

    4. At desired time points after injection, monitor the health of the larvae before beginning additional analysis.

      Note: Larvae should show no overt signs of change and should be indistinguishable from non- injected stage-matched controls. Healthy larvae will have a consistent rhythmic heartbeat with apparent blood flow, have intermittent bursts of movement, and be straight bodied.

Data analysis

Examples of data analysis using this method can be found in previous publications (Earley et al., 2018; Yang et al., 2020) where the experiments have used brain or intravenous microinjection of LPS or bacteria to examine various types of immune response. Downstream analyses include determining changes in expression of immune response genes (see Figure 5 in Earley et al., 2018 and Figure 5 in Yang et al., 2020), dynamic cellular behaviors and mobilization of macrophages (see Figure 1 in Yang et al., 2020), and routes of molecular drainage from the brain to the periphery (see Figure 2 in Yang et al., 2020).

Recipes

  1. 50× PTU stock solution (1L)

    Add 1.5 g N-Phenylthiourea (PTU) to 1 L of distilled water and stir overnight at room temperature.

    Store at -20 °C.

    Note: To inhibit pigmentation in developing embryos use at a 1× concentration in fish water for a final concentration of 0.003% PTU and replace daily. The liquid 50× stock can be stored at room temperature for several months.

  2. 25× Tricaine stock solution (100 ml)

    Add 400 mg tricaine powder (3-amino benzoic acid ethyl ester) to 100 ml distilled water.

    Store at -20 °C.

    Note: To administer tricaine as an anaesthetic use at 0.5×-1× concentration. The liquid 25× stock can be stored at room temperature for several months.

  3. 1.5% low melt agarose (100 ml)

    Add 1.5 g low melt agarose powder to 100 ml distilled water.

    Heat for 20 s to less than 1 min in microwave until solution is clear and fluid.

    Store at room temperature in a glass bottle with a lid and can be repeatedly re-used by re-melting the agarose in the microwave.

    Note: To prevent liquid from boiling over, stand nearby to monitor. Multiple rounds of heating can result in evaporation of liquid and increase the gel percentage.

Acknowledgments

This protocol accompanies the publications (Earley et al., 2018; Yang et al., 2020). The work was funded by NIH NIGMS (Grant Number 1R35GM124719 to C.E.S.)

Competing interests

The authors declare no competing interests.

Ethics

Animal experimentation: This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All of the animals were handled according to approved institutional animal care and use committee (IACUC) protocols (#16-160 and #19-132) of the UNC Chapel Hill.

References

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  2. Davis, J. M., Clay, H., Lewis, J. L., Ghori, N., Herbomel, P. and Ramakrishnan, L. (2002). Real-Time Visualization of Mycobacterium-Macrophage Interactions Leading to Initiation of Granuloma Formation in Zebrafish Embryos. Immunity 17(6): 693-702.
  3. Earley, A. M., Dixon, C. T. and Shiau, C. E. (2018). Genetic analysis of zebrafish homologs of human FOXQ1, foxq1a and foxq1b, in innate immune cell development and bacterial host response. PloS One 13(3): e0194207.
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简介

[摘要]创建一个健壮和控制感染模型是势在必行用于学习的先天免疫应答。凭借特殊的斑马鱼模型系统的优势,比如光学透明性,前子宫发展,以及大离合器的尺寸,允许小号的了的,其产生方法的发展是一致和可重复的 结果。我们通过将细菌颗粒或活细菌显微注射到幼虫斑马鱼中创建了一个强大的激活先天免疫的模型,这与以前的研究在很大程度上将此类操作限制在斑马鱼的胚胎阶段不同。在幼虫阶段局部或全身引入刺激的能力提供了显着的优势,可检查更成熟组织中的宿主反应,并在较老的幼虫阶段询问适应性免疫的可能性。这个协议描述2种显微注射的不同模式以引入脂多糖(LPS)或细菌到活幼虫斑马鱼:一个本地化到的脑,和另一个为对经由尾静脉血流丛。


图形摘要:

示意性示出了两种不同的模式的幼虫斑马鱼显微注射,无论是在脑实质或在血流中静脉内。ř eagents引入斑马鱼,以评估免疫应答中作为在协议中描述的“注入组件”被描绘。

[背景]感染过程中复杂的相互作用需要使用体内动物模型来充分了解病原体与其宿主之间的动态相互作用。研究此现象需要病原体传递的受控和可靠方法。斑马鱼已被用作研究多种病原体免疫应答的模型(Menudier等,1996; Davis等,2002; Neely等,2002; Prouty等,2003; van der Sar等)等人,2003年;O'Toole等人,2004年; Phelan等人,2005年; Phelps和Neely,2005年; Pressley等人,2005年),因为它具有进化保守的先天免疫系统(Herbomel等人,1999年; Traver等人,2003; Trede等人,2004),光学透明性,大的胚离合大小,遗传易处理性和体内成像能力(Kimmel,1989; Kimmel等人,198 8和1995; Sullivan和Kim,2008 ;等)。Kanther和Rawls,2010年)。以前的感染方案是通过将鱼直接添加到鱼水中的方法将鱼浸入病原体中(Davis等,2002; O'Toole等,2004; Prouty等,2003),或者将细菌注入到轴静脉或后脑室中。受精后28 h的早期胚胎(hpf)(van der Sar et al 。,2003),或受精后3-6天(dpf)在后期将LPS注入幼虫斑马鱼中,诱导卵黄致命的全身免疫反应(Yang等,2014)。从以前发表的方法扩展,我们开发了一种将LPS或细菌在幼虫斑马鱼中显微注射的协议,可以直接注射到脑实质或血液循环中,以引起从4 dpf幼虫开始的强大的先天免疫应答。由于适应性免疫直到斑马鱼受精后约4周才开始(Davis et al 。,2002),因此我们可以利用斑马鱼的幼体早期阶段来研究与适应性免疫无关的特定先天免疫功能。与荧光标记的葡聚糖,或荧光标记免疫激活剂直接注射免疫激活剂联合注射允许小号的成功注入的快速视觉验证,以及基于报告的吞噬作用的巨噬细胞反应的后续标记。该协议描述了两种具有不同目标部位的显微注射模式:首先,用于在整个血流中进行全身分布的尾静脉丛,其次,是短暂地将注射物质定位在大脑中但随后排入循环中的大脑皮层(Yang等人,2020)。尽管我们描述我们的协议3-5 DPF幼虫,这些方法适用于至少好以后幼虫阶段10 DPF(杨等人。,2020)。

关键字:巨噬细胞, 显微注射, 斑马鱼, 炎症, 感染, 免疫学, 大脑, 循环

材料和试剂

1.聚苯乙烯培养皿(VWR,目录号:25384-342)         

2. 7.5毫升移液器(VWR,目录号:414004-005)         

3.薄壁玻璃毛细管4,带细丝,外径1.5 mm(World Precision仪器,目录号:TW150F-4)         

4.大肠杆菌(任何菌株)         

5.大肠杆菌O111:B4的脂多糖(Sigma - Aldrich,目录号:L3024)(储存在-20°C)         

6.大肠杆菌055:B5血清脂多糖,Alexa Fluor TM 594偶联物(Thermo Fisher Scientific,Invitrogen,目录号:L23353)(储存在-80 °C并避光)         

7. Dextran,Alexa Fluor TM 568(Thermo Fisher Scientific,Invitrogen,目录号:D-22912)(储存在-20°C并避光)         

8. P hosphate缓冲盐水(PBS ),pH 7.4的         

9. 1.5%低熔点琼脂糖(Fisher Scientific,IBI Scientific,目录号:50-550-455)(请参阅食谱)         

10.三卡因(3-氨基苯甲酸乙酯)(Sigma-Aldrich,目录号:A-5040)(在室温下保存)     

注意:小号当制成25撕在-20℃ ×三卡因溶液(见配方)。


11. PTU(N-苯基硫脲)(Sigma-Aldrich,目录号:P7629)(在室温下存储)     

注意:小号当制成50撕在-20℃ × PTU溶液(见配方)。


设备


1.可编程卧式移液器拔轮器(World Precision Instruments,目录号:PUL-1000)         

2.气动PicoPump (World Precision仪器,目录号:SYS-PV820)         

3.荧光体视显微镜,通过16:5:1变焦光学元件完全复消色差校正(Leica,型号:M165 MC)         

4.孵化器(Benchmark Scientific,型号:H2200-H)         

5.载物台测微计,1 × 0.01 mm(AmScope,目录号:MR095)         

6.磁力架(世界精密仪器公司,目录号:M1)         

7.手动微操纵器(World Precision Instruments,目录号:M3301)         

8.钢制底板,10磅(World Precision仪器,目录号:5052)         

9. PicoNozzle套件v2(World Precision仪器,目录号:5430-ALL)         

10. Dumont#55镊子(Fine Scientific Tools,目录号:11295-51)     



程序


A.斑马鱼的胚胎和幼体饲养         

            用加有0.003%PTU的高压灭菌鱼水在培养皿中保持斑马鱼的温度,并在28.5°C下孵育。
对于一般性问题,包括Avdesh等人在内的其他地方对鱼水和常规斑马鱼饲养的组成进行了很好的描述。(2012年)。


每天用含有0.003%PTU的淡水代替水,并监控健康状况。
当胚胎达到所需的幼虫阶段(例如4 dpf)时,开始实验。


B.注射混合物的准备         

            Ë 。补充有荧光右旋糖酐的大肠杆菌:准备3 ml过夜培养的单个菌落。离心培养物在3,000rpm下1分钟,取出上清液并重新悬浮在500微升的1 × PBS(pH7.4)中。这应该是大约1.6 × 10 6 CFU /微升。加入1微升的5纳克/ NL荧光葡聚糖。在使用前,用手指轻拂试管并向下旋转。
            LPS补充有荧光葡聚糖:混合9微升的LPS以5ng / NL从大肠埃希氏大肠杆菌O111:B4和1μl的1:10稀释的5纳克/ NL荧光标记的葡聚糖,以使1的最终稀释:100补充荧光右旋糖酐。在使用前,用手指轻拂试管以使其混合并向下旋转。
LPS直接偶联于荧光分子(例如,Alexa的594)- ü直接瑟在5毫微克/ NL。
            使用补充有荧光右旋糖酐的超纯水或高压灭菌水控制车辆注射 -米IX 9微升的水与1微升的一个1:10稀释的5纳克/ NL荧光标记的葡聚糖。在使用前,用手指轻拂试管以使其混合并向下旋转。


C.针头和微型注射器的设置         

使用可编程水平移液器或类似设备将微量注射针从玻璃毛细管中拉出(表1)。




表1.玻璃毛细管拉拔的4步操作规程


将约3μl的注射溶液装入针头。
注意:除非使用非常细的移液器吸头,否则溶液将残留在针头的嘴中。要将溶液推到针尖,请用力将针牢牢握在中指和拇指之间,并以向下运动的方式轻拂手腕(请参见视频1)。重复直到液体到达针头的狭窄尖端。




视频1.加载注入混合物到一个显微注射针


转进针显微,并阐明了的的方式。


D.安装斑马鱼以进行显微注射(请参见视频2)         





视频2.分步演示用于显微注射的幼虫安装


使用塑料移液管将幼虫运输到干净的培养皿盖的中心,并尽可能多地除去水。
注意:之所以使用陪替氏培养皿盖是因为盖的侧面具有较低的轮廓,这允许更大的自由度将针定位到所需的目标。可以一次安装多个幼虫;对于脑部注射,我们可以一次安装20至30头以上的斑马鱼幼虫。


在微波中加热1.5%低熔点琼脂糖(固体形式),使其熔融成液体形式。
注:约20秒至小于1分钟时需要熔体100毫升琼脂糖。低熔点琼脂糖加热非常快,因此您需要站在旁边监视加热情况,以防止沸腾。


使用移液器收集少量的低熔点琼脂糖。
微波加热后,立即琼脂糖会非常热。如果你看到蒸汽/冷凝内的吸管,然后将琼脂糖是太炎热而将燃烧的幼虫。仔细触摸琼脂糖所在的移液器外部,以定期监视琼脂糖的温度。琼脂糖应温暖接触,而不要热,并保持液体流动。
低熔点琼脂糖在37 °C时仍会保持液态,并在25 °C以下迅速凝固。旨在在30 °C和40 °C之间的温度下使用琼脂糖。保持琼脂糖加热块上在60 ℃下将保持在流体状态中的溶液为一个的时间较长期间你上安装幼虫工作。


可以通过上下几次将热的琼脂糖移到干净的表面(例如另一个培养皿)上来加快冷却琼脂糖的速度(请参见视频2)。
            形成环绕幼虫一圈与琼脂糖注射进去较小的同心圆运动,直到将琼脂糖触及的幼虫。混合的琼脂糖用的幼虫通过使用吸管轻轻摇动在一起或吹打他们上下1-2倍(见视频2)。
注意:为了便于注射,目的是将幼虫包埋在薄薄的琼脂糖层中,以使细小玻璃针容易穿透凝胶。使用过多的琼脂糖会使针的定位更加困难,并导致玻璃针弯曲。


使用镊子定向幼虫。
Ť他是最敏感的时间的步骤,因为幼虫必须正确定位在琼脂糖重新固化(1-2分钟)之前。根据所需的注射部位,使用细镊子快速定向每个幼虫,但不必担心确切的定向或身体对准。小心不要戳戳它们以伤害幼虫,而应使用周围的琼脂糖轻轻推动它们并将其移动到位(请参见视频2)。
对于脑部和静脉注射,将幼虫分别置于其背侧或腹侧。
等待琼脂糖冷却并固化,然后再开始注射。
            在琼脂糖上添加少量鱼类系统水,以使幼虫在水中保持健康,并在手术过程中保持健康。


E.建立气动微量和校准您的针注射         

            在操作之前,请阅读制造商对微针拔针器和微注射器的建议和说明。
            打开上PicoPump和开放空气阀。的输出压力应被设定至20磅。在放置针头之前,请检查是否感觉到空气从针头支架(PicoNozzle套件v2)中喷出。
            插入拉出的玻璃针。准备额外的针头,以防万一它们断裂或需要重新校准。
            通过显微镜观察,通过调节固定在固定您的针的钢质底板上的磁性支架上的手动微操纵器,将针尖对准视野的中央。
            用细镊子来轻轻地打破过小的控制量的的玻璃针。的目的是要打破的最小量的所述针,以允许所述最小量的液体喷射出去(参见图1 )。




图1 。使用注射针的校准一个微米。为确保注入量均匀,请使用千分尺测量注入气泡量的直径。100μm的注入气泡直径等于0.5 nl 。


            按下PicoPump脚踏板,以查看在针尖上是否可见大量的液体。如果没有液体,请继续小心地从针头上夹下小碎片,直到看到少量液体为止。
            使用千分尺校准针孔的大小(请参见图1 )。
调整由碎裂的玻璃针的开口大小关的下递增结束立体镜而你测试如何多少体积被排出后的每个球通过在空气中在右边的针尖估计球形液滴尺寸以上的千分尺。
力求使球形滴的直径在千分尺上约为100μm,以使PicoPump脚踏板的每个脚踏产生0.5 nl的体积(请参见图1 )。
注意:为防止注射部位出现并发症,请最小化针头的尺寸。W¯¯ Ë建议使用较小的针和辖荷兰国际集团1个NL注射2个踢。


可以通过标准公式确定球体体积(V = 3 )来确定针上液滴的体积(V ),其中r是液滴的半径。


F.脑显微注射         

            在荧光立体显微镜下进行显微注射,以监测和验证每次成功的荧光注射。所述安装在幼虫低熔点琼脂糖将需要被定向与脑侧开针访问(参见图2 )。
将针头直接放在大脑顶盖上方(请参见图2 )。
使用显微操纵器用针慢慢刺穿皮肤。
注:力的的穿刺可驱动的针进一步进入的大脑比期望的。向后拉动针头,直到针头只刺穿大脑。


按下PicoPump脚踏板两次以弹出1 nl。
屏幕成功注射通过可视化在大脑中顶盖荧光在注射部位(参见图2乙)。
注意:注射液扩散到后脑室是正常的。使用镊子将幼虫从琼脂糖中取出,取出未正确注射的幼虫。




图2 。脑部和静脉微注射部位。A.红色箭头指向右保护层中的大脑注射部位,白色框勾勒出3 dpf处幼虫脑的右保护层区域。B.荧光葡聚糖注射后30秒离子进入脑顶盖(红色箭头),该荧光示踪剂可以容易地观察到分散入脑脑室和进入椎管(红色箭头)。C.红色箭头指向位于背主动脉下方(尾部红色箭头)且靠近泌尿生殖器开口(底部红色箭头)的尾静脉丛的静脉注射部位。D.全身图像30秒后静脉注射示出在整个荧光葡聚糖信号身体vascula TURE 。红色箭头小号点在血管头部和躯干的可见荧光葡聚糖。白色虚线框,EE中所示的区域。静脉注射后,心脏(红色圆圈)立即充满了右旋糖酐荧光信号。红色箭头指向the脉管系统中的荧光信号。


G.静脉显微注射         

在荧光立体镜下进行显微注射,以监测和验证每次成功的荧光注射(参见图2 )。
            您在安装幼虫低熔点琼脂糖将需要被定向与腹侧侧面向上到访问的蛋黄延伸侧的所述尾到注入到所述尾静脉丛(参见图2 Ç )。
注意:这是最能够可视化和瞄准针进入组织中的薄带有效尾静脉丛,这立即后到结束是体壁的最腹侧的所述蛋黄延伸,其中连续循环可以是容易在尾静脉中观察到(见图2 C )。


将针头直接放置在尾静脉丛附近靠近泌尿生殖器开口处(参见图2 C )。
使用显微操纵器用针慢慢刺穿皮肤。
注意:穿刺的力可以驱动针头穿过静脉并进入肌肉。向后拉动针头直到其进入静脉。


按下PicoPump脚踏板两次以弹出1 nl。
            一个成功的注射后,荧光的直接检测将在心脏发生(图2E)和从那里的注射物质将被广泛分发整个所述本体脉管系统是完全可见秒内1分钟的后喷射(参见图2 d - Ë )。
注意:取出所有未正确注射的幼虫。静脉注射比脑注射需要更多的技术实践,因为目标组织(尾静脉神经丛)远比顶盖薄且面积小。


H.注射后幼虫的恢复       

通过从琼脂糖中去除注入的胚胎来恢复它们。
一种。使用镊子的侧面在头下方开始的琼脂糖中打断。 

b。沿身体拖动镊子,直到到达尾巴的尖端。 

C。幼虫应该能够容易地蠕动并在凝胶上方的鱼水中游泳。 

使用移液管将注入的幼虫放入干净的陪替氏培养皿中,并用新鲜鱼水和PTU补充。
将幼虫放入28.5°C的培养箱中进行恢复。
注射后在所需的时间点,在开始其他分析之前,监测幼虫的健康状况。
注意:幼虫不应显示明显的变化迹象,并且应与未注射的阶段匹配对照区分开。ħ ealthy幼虫将具有明显的血流量一致的韵律心跳,具有运动的间歇脉冲串,并且是直浓郁。


数据分析


使用这种方法进行数据分析的例子可以在以前的出版物中找到(Earley等人,2018; Yang等人,2020),其中实验使用脑或静脉内显微注射LPS或细菌来检查各种类型的免疫反应。下游分析包括确定免疫应答基因表达的变化(参见Earley等,2018的图5和Yang等,2020的图5 ),动态细胞行为和巨噬细胞的动员(参见Yang等的图1)。 (2020年),以及从大脑到外围的分子排泄途径(请参见Yang等人,2020年的图2 )。


菜谱


1. 50 × PTU储备溶液(1升)     

将1.5 g N-苯基硫脲(PTU)添加到1 L蒸馏水中,并在室温下搅拌过夜。


储存在-20°C 。


注:要在发展中抑制色素沉着的胚胎使用在一个1 ×浓度的鱼水为一个最终浓度的0.003%PTU和更换日常。50 ×储液可以在室温下保存几个月。


2. 25 × Tricaine储备溶液(100 ml)     

将400毫克三卡因粉末(3-氨基苯甲酸乙酯)加到100毫升蒸馏水中。


储存在-20°C 。


注意:以0.5 × -1 ×的浓度将三卡因作为麻醉剂给药。25 ×储液可以在室温下保存几个月。


3. 1.5%低熔点琼脂糖(100毫升)     

添加1.5克低熔融琼脂糖粉末到100 ml的蒸馏水水。


在微波中加热20秒钟至不到1分钟,直到溶液澄清并呈流体状。


室温下保存在带盖玻璃瓶中,可以通过在微波炉中重新融化琼脂糖来重复使用。


注意:为防止液体沸腾,请站在显示器旁边。多个轮的加热可导致在的液体蒸发,增加凝胶的百分比。


致谢


该协议随出版物一起发布(Earley等,2018; Yang等,2020)。这项工作是由NIH NIGMS资助的(授予CES的批准号1R35GM124719)


利益争夺


作者宣称没有利益冲突。


伦理


动物实验:严格按照《美国国立卫生研究院实验动物的护理和使用指南》中的建议进行这项研究。所有动物均根据UNC教堂山的认可机构动物护理和使用委员会(IACUC)规程(#16-160和#19-132)进行处理。


参考


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Copyright Rojas and Shiau. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Rojas, A. M. and Shiau, C. E. (2021). Brain-localized and Intravenous Microinjections in the Larval Zebrafish to Assess Innate Immune Response. Bio-protocol 11(7): e3978. DOI: 10.21769/BioProtoc.3978.
  2. Yang, L., Jiménez, J. A., Earley, A. M., Hamlin, V., Kwon, V., Dixon, C. T. and Shiau, C. E. (2020). Drainage of inflammatory macromolecules from the brain to periphery targets the liver for macrophage infiltration. eLife 9: e58191.
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