Key words 关键词

1 Introduction 1 引言

Craniofacial defects, affecting approximately one in a thousand live births, are one of the most common congenital disabilities in humans. Mouth-facial clefts, one of these defects, are encountered in both developed and developing countries. Although craniofacial defects are not usually life-threatening, they can seriously reduce a person’s quality of life by causing problems with eating, hearing, speech, and breathing. It has been shown that genetic and environmental factors may play a role in the development of craniofacial defects encountered in the embryonic period in vertebrates [1, 2].
颅面缺陷影响大约千分之一的活产，是人类最常见的先天性残疾之一。口面裂是这些缺陷之一，在发达国家和发展中国家都有。虽然颅面缺陷通常不会危及生命，但它们会导致进食、听力、言语和呼吸问题，从而严重降低一个人的生活质量。研究表明，遗传和环境因素可能在脊椎动物胚胎期遇到的颅面缺损的发展中发挥作用[1,2]。

Zebrafish (Danio rerio), a model organism increasingly used in toxicological research in recent years, is very suitable for evaluating the effects of different compounds on bone and cartilage structures. The process of formation of the skeletal system in the early stages of development is highly conserved across species, and zebrafish are therefore considered suitable for studying skeletal malformations in humans.
斑马鱼（Danio rerio）是近年来毒理学研究中越来越多地使用的模式生物，非常适合评估不同化合物对骨骼和软骨结构的影响。在发育的早期阶段，骨骼系统的形成过程在物种中是高度保守的，因此斑马鱼被认为适合研究人类的骨骼畸形。

The use of zebrafish as a model organism has some particular advantages. Chief among these is that embryos are transparent in the early stages of their development and are excluded from the scope of animal experimentation legislation until 120 h post-fertilization (hpf) (5 days after fertilization-5 dpf). The chondrocranium is a cartilaginous skeletal component of the cranial base and also known as the cartilaginous neurocranium, which develops to enclose the rapidly expanding embryonic brain. Research on zebrafish craniofacial development has dramatically improved our understanding of cartilage and bone growth in teleosts. In the zebrafish embryos, chondrogenesis is already established by embryonic day 2, and supporting cartilage elements are detectable at hatching (72 hpf) [3]. The development of the chondrocranium follows the regular vertebrate pattern, with more anterior cartilage elements deriving from the neural crest and more posterior cartilage components having a mixed origin, likely with a mesodermal involvement [4].
使用斑马鱼作为模式生物具有一些特殊的优势。其中最主要的是胚胎在其发育的早期阶段是透明的，并且在受精后 120 小时 （HPF） （受精后 5 天 - 5 DPF） 之前被排除在动物实验立法的范围之外。软骨颅骨是颅底的软骨骨骼成分，也称为软骨神经颅骨，其发育以包围快速扩张的胚胎大脑。对斑马鱼颅面发育的研究极大地提高了我们对硬骨软骨和骨骼生长的理解。在斑马鱼胚胎中，软骨生成在胚胎第2天就已经建立，并且在孵化时（72 hpf）可以检测到支持软骨元件[3]。软骨颅骨的发育遵循常规的脊椎动物模式，更多的前软骨成分来自神经嵴，更多的后软骨成分具有混合起源，可能与中胚层受累[4]。

The most significant cartilaginous part of the ventral hyoid arch is the bilaterally paired pharyngeal arch cartilage, ceratohyal cartilage. Numerous signaling pathways are involved in the normal development of the zebrafish craniofacial region. The transcriptional cascade that suppresses follistatin A comprises Runx3, Egr1, and Sox9b, originating in the pharyngeal endoderm. The cranial neural crest cell-derived chondrocytes are stimulated to expand and multiply by this cascade [5]. Immature chondrocytes near the medial epiphysis of the ceratohyal cartilage consist of a group of cells that divide, intercalate, and lengthen the cartilage during normal development. This process is known as a convergent extension [6, 7].
腹侧舌骨弓最重要的软骨部分是双侧成对的咽弓软骨，即角膜软骨。许多信号通路参与斑马鱼颅面区域的正常发育。抑制卵泡抑素 A 的转录级联反应包括 Runx3、Egr1 和 Sox9b，起源于咽内胚层。颅神经嵴细胞衍生的软骨细胞被这种级联刺激扩增[5]。角膜软骨内侧骨骺附近的未成熟软骨细胞由一组细胞组成，这些细胞在正常发育过程中分裂、插入和延长软骨。这个过程被称为收敛扩展[6,7]。

The cranial skeleton is easily visible in 72–120 hpf embryos, especially after cartilage is stained using alcian blue [8, 9]. Accordingly, Meckel’s and ceratohyal cartilages can be visualized by staining them with Alcian Blue. Various distances and angles are evaluated to optimize the quantitative analysis of cranial malformations in zebrafish after exposure to various toxic agents [1].
在72-120 hpf的胚胎中很容易看到颅骨，特别是在软骨用阿尔新蓝染色后[8,9]。因此，梅克尔软骨和角膜软骨可以通过用阿尔新蓝染色来可视化。评估各种距离和角度，以优化斑马鱼暴露于各种有毒物质后颅骨畸形的定量分析[1]。

Specific cranial length and structures determined by Alcian blue staining are mainly palatoquadral cartilage (PQ) and Meckel’s angle, ceratohyal cartilage (CH) angle, PQ length, and CH to Meckel length (Fig. 1); and ceratohyal cartilage length (CCL), intraocular distance (ID), and lower jaw length (LJL) (Fig. 2).
阿尔新蓝染色确定的具体颅长和结构主要是腭方软骨（PQ）和梅克尔角、角软骨（CH）角、PQ长度和CH到梅克尔长度（图1）;和神经软骨长度 （CCL）、眼内距离 （ID） 和下颌长度 （LJL）（图 2）。

Fig. 1 图1
A 6-part schematic illustration of the specific cranial length and structures of larva in A to F marking the width, C H angle, P Q Meckel's angle, M-C H length, and P Q length.

Demonstration of specific cranial length and structures (a): Larva imaged at the end of the protocol stages; (b): Displaying palatoquadral cartilage (PQ) and Meckel’s angle, ceratohyal cartilage (CH) angle and width on the schematized larva; (c): Displaying PQ length and M-CH length on the schematized larva; (d): Displaying the width measurement; (e): Displaying palatoquadral cartilage (PQ) and Meckel’s angle and CH angle; (f): Displaying PQ length and M-CH length on the schematized larva
特定颅骨长度和结构的演示（a）：在协议阶段结束时对幼虫进行成像;（b）：显示图式化幼虫的腭方软骨（PQ）和Meckel角、角膜软骨（CH）角和宽度;（c）：显示图式化幼虫的PQ长度和M-CH长度;（d）：显示宽度测量值;（e）：显示腭方软骨 （PQ） 和 Meckel 角和 CH 角;（f）：显示图式化幼虫的PQ长度和M-CH长度

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Fig. 2 图2
A 3-part schematic illustration of the length and structures of larva marking the C C L, I D, and L J L.

Demonstration of ceratohyal cartilage length (CCL), Intraocular Distance (ID) and Lower jaw length (LJL)
角膜软骨长度 （CCL）、眼内距离 （ID） 和下颌长度 （LJL） 的演示

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2 Materials 2 材料

All solutions should be made with analytical-grade chemicals and ultrapure water, created by filtering deionized water to a sensitivity of 18 million ohm-cm (18 megohm) at 25 °C. Unless otherwise stated, all solutions should be prepared and kept at room temperature.
所有溶液都应使用分析级化学品和超纯水制成，这些水是通过在25°C下将去离子水过滤至1800万欧姆（18兆欧姆）的灵敏度而制成的。 除非另有说明，否则所有溶液都应制备并保存在室温下。

2.1 Required Equipment 2.1 所需设备

  1. 1.

    Beakers. 烧杯。

  2. 2.

    Borosilicate sterile petri dishes.
    硼硅酸盐无菌培养皿。

  3. 3.

    Elga Option-Q Ultra water purifier.
    Elga Option-Q Ultra 净水器。

  4. 4.

    pH meter. pH计。

  5. 5.

    Micropipette and tips. 微量移液器和吸头。

  6. 6.

    Incubator. 孵化器。

  7. 7.

    Powder-free gloves. 无粉手套。

  8. 8.

    Analytical balance. 分析平衡。

  9. 9.

    Volumetric flasks. 容量瓶。

  10. 10.

    Vortex mixer. 涡旋混合器。

2.2 Required Software 2.2 所需软件

  1. 1.

    Image Software for image collection.
    用于图像收集的图像软件。

2.3 Required Solutions 2.3 所需解决方案

  1. 1.

    E3 Medium: For 1 L, add 5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, and 0.33 mM MgSO4. Adjust pH to be around 7.2.
    E3培养基：对于1L，加入5mM NaCl，0.17mM KCl，0.33mM CaCl 2 和0.33mM MgSO 4 。将 pH 值调节至 7.2 左右。

  2. 2.

    PBS: For 1 L, add 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4.
    PBS：对于 1 L，加入 137 mM NaCl、2.7 mM KCl、10 mM Na 2 HPO 4 和 1.8 mM KH 2 PO 4 。

  3. 3.

    PBT: For 1 L PBS, add 1 g of Tween® 20 detergent.
    PBT：对于 1 L PBS，加入 1 克 Tween ® 20 洗涤剂。

  4. 4.

    60 mM MgCl2: Add 571 mg MgCl2 in 100 mL distilled water.
    60 mM MgCl 2 ： 2 在 100 mL 蒸馏水中加入 571 mg MgCl。

  5. 5.

    10 mM MgCl2: Add 203 mg MgCl2 in 100 mL distilled water (see Note 1).
    10 mM MgCl 2 ： 2 在 100 mL 蒸馏水中加入 203 mg MgCl（见注 1）。

  6. 6.

    Ethanol (25%): Add 25 mL of ethanol (96–100% molecular biology grade) and 75 mL distilled water.
    乙醇 （25%）：加入 25 mL 乙醇（96-100% 分子生物学级）和 75 mL 蒸馏水。

  7. 7.

    Ethanol (50%): Add 50 mL ethanol and 50 mL distilled water.
    乙醇 （50%）：加入 50 mL 乙醇和 50 mL 蒸馏水。

  8. 8.

    Ethanol (70%): Add 70 mL ethanol and 30 mL distilled water.
    乙醇 （70%）：加入 70 mL 乙醇和 30 mL 蒸馏水。

  9. 9.

    Ethanol (80%): Add 80 mL ethanol and 20 mL distilled water.
    乙醇 （80%）：加入 80 mL 乙醇和 20 mL 蒸馏水。

  10. 10.

    Glycerol (25%): Add 30 mL of glycerol (99% pure) in 70 mL of distilled water.
    甘油 （25%）：在 70 mL 蒸馏水中加入 30 mL 甘油（纯度为 99%）。

  11. 11.

    Glycerol (50%): Add 50 mL of glycerol in 50 mL of distilled water.
    甘油 （50%）：在 50 mL 蒸馏水中加入 50 mL 甘油。

  12. 12.

    Glycerol (70%): Add 70 mL of glycerol in 30 mL of distilled water (see Note 2).
    甘油 （70%）：在 30 mL 蒸馏水中加入 70 mL 甘油（见注 2）。

  13. 13.

    10% Tricaine-S: For 100 mL solution, add 1 g of Tricaine-S and 2 g of sodium bicarbonate into 80 mL of distilled water and mix until dissolved. The volume is completed 100 mL with distilled water [10, 11] (see Note 3).
    10% Tricaine-S：对于 100 mL 溶液，将 1 g Tricaine-S 和 2 g 碳酸氢钠加入 80 mL 蒸馏水中，混合直至溶解。用蒸馏水[10,11]完成体积100mL（见注3）。

  14. 14.

    H2O2 (3%): Since the concentration of stock hydrogen peroxide is 30%, 1× is taken from the stock to obtain 3% H2O2 and is completed with distilled water to 10× volume. For a 100 mL solution, dilute 10× by pipetting 10 mL of 30% H2O2 to 90 mL distilled water.
    2 H O 2 （3%）：由于原液过氧化氢的浓度为30%，因此从原料中取1×得到3%的H 2 O 2 ，并用蒸馏水完成至10×体积。对于 100 mL 溶液，将 10 mL 30% H 2 O 2 移液至 90 mL 蒸馏水中，稀释 10×。

  15. 15.

    KOH (0.5%): Add 0.5 g of potassium hydroxide powder in 100 mL of distilled water and dissolve.
    KOH（0.5%）：在100mL蒸馏水中加入0.5g氢氧化钾粉末并溶解。

  16. 16.

    KOH (0.1%): Add 0.1 g of potassium hydroxide powder in 100 mL distilled water and dissolve.
    KOH（0.1%）：在100mL蒸馏水中加入0.1g氢氧化钾粉末并溶解。

  17. 17.

    0.01% Alcian Blue: The 0.01% solution represents the amount of 10 mg of substance in 100 mL of solvent. The solution is prepared by mixing and dissolving 10 mg Alcian Blue powder and 60 mM MgCl2 solution dissolved in 100 mL 70% ethanol.
    0.01%阿尔新蓝：0.01%溶液代表100mL溶剂中10mg物质的量。通过混合并溶解10mg阿尔新蓝粉末和溶解在100mL 70%乙醇中的60mM MgCl 2 溶液来制备溶液。

3 Methods 3 方法

Alcian Blue is a cationic dye from the family of phthalocyanines, the synthetic equivalents of porphyrins having nitrogen-aromatic rings encircling a core atom. In particular, when the pH is acidic (pH 2.2), these compounds are essential molecules that attach to the acid sites of their targets and result in the production of a saline compound. The existence of a copper atom in the molecule gives Alcian Blue its blue color.
阿尔新蓝是酞菁家族的一种阳离子染料，酞菁是卟啉的合成等价物，具有围绕核心原子的氮芳环。特别是，当pH值为酸性（pH 2.2）时，这些化合物是附着在其目标的酸位点上并导致产生盐水化合物的必需分子。分子中铜原子的存在使阿尔新蓝呈蓝色。

In connective tissues like cartilage and bone, the cells are encased in a dense extracellular matrix that is generally stiff. Most of the proteoglycans and glycoproteins make up the extracellular matrix, which is hydrated and viscous and contains embedded collagen and elastin fibers. Extracellular glycoproteins typically bind a significant percentage of glycosaminoglycans. Glycoproteins are molecules containing negatively charged sulfate groups that bind both water and cations, creating a hydrated and gelatinous matrix. They also contain hydrophilic carboxyl groups, which attract water. The four most significant glycoprotein forms are chondroitin sulfate, hyaluronic acid, heparin sulfate, and keratin sulfate, found in bone and cartilage tissue. The selective binding of Alcian Blue to acidic glycoproteins in an acidic solution (pH 2.2) results in the staining of zebrafish cartilage. GAGs with acid sulfate groups exhibit a particular affinity for AB, which causes them to become blue.
在软骨和骨骼等结缔组织中，细胞被包裹在通常坚硬的致密细胞外基质中。大多数蛋白聚糖和糖蛋白构成细胞外基质，细胞外基质水合且粘稠，并含有嵌入的胶原蛋白和弹性蛋白纤维。细胞外糖蛋白通常结合相当大比例的糖胺聚糖。糖蛋白是含有带负电荷的硫酸根基团的分子，这些基团结合水和阳离子，形成水合和凝胶状基质。它们还含有亲水性羧基，可吸引水。四种最重要的糖蛋白形式是硫酸软骨素、透明质酸、硫酸肝素和硫酸角蛋白，存在于骨骼和软骨组织中。阿尔新蓝在酸性溶液（pH 2.2）中与酸性糖蛋白的选择性结合导致斑马鱼软骨染色。具有酸性硫酸基团的 GAG 对 AB 表现出特殊的亲和力，这导致它们变成蓝色。

Alcian Blue preparations can visualize individual cartilage as early as 2 days after fertilization (dpf). We used 72 hpf (hours post fertilization) zebrafish embryos in this protocol. To determine teratogenicity, zebrafish embryos exposed to different substances are compared with healthy embryos in the control group in terms of cartilage staining. The main objective is to identify craniofacial cartilaginous structures in zebrafish embryos.
阿尔新蓝制剂最早可以在受精后 2 天 （dpf） 看到单个软骨。在该协议中，我们使用了72 hpf（受精后数小时）斑马鱼胚胎。为了确定致畸性，将暴露于不同物质的斑马鱼胚胎与对照组的健康胚胎进行软骨染色进行比较。主要目的是鉴定斑马鱼胚胎中的颅面软骨结构。

The staining method was modified following Staal et al., Schilling et al., and Dingerkus and Uhler’s studies [8, 12, 13].
根据Staal等人、Schilling等人以及Dingerkus和Uhler的研究[8,12,13]修改了染色方法。

3.1 Protocol Stages 3.1 协议阶段

  1. 1.

    The embryos should be thoroughly washed in embryo medium (E3) or phosphate-buffered saline with 0.1% Tween-20 (PBT) (see Note 4).
    胚胎应在胚胎培养基（E3）或含0.1%吐温-20（PBT）的磷酸盐缓冲盐水中彻底洗涤（见注4）。

  2. 2.

    Anesthetize the embryos with Tricaine-S. Fix for 2 h in 4% paraformaldehyde (PFA) at room temperature.
    用Tricaine-S麻醉胚胎。在室温下在4%多聚甲醛（PFA）中固定2小时。

  3. 3.

    Then, the fixed embryos are washed three times with PBS for 5 min.
    然后，将固定胚胎用PBS洗涤3次5分钟。

  4. 4.

    Bleach the embryos until the eyes are translucent enough in 3% H2O2 and 0.5% KOH for 35 min. You can see the steps up to here in Fig. 3.
    漂白胚胎，直到眼睛在3%H 2 O 2 和0.5%KOH中足够半透明，持续35分钟。您可以在图 3 中看到到此处的步骤。

  5. 5.

    Wash and rinse the embryos three times with PBS for 5 min.
    用PBS清洗并冲洗胚胎三次，持续5分钟。

  6. 6.

    Embryos are incubated at 4 °C overnight in 0.01% Alcian Blue dissolved in 60 mM MgCl2 and 70% ethanol in the dark (see Notes 5 and 6).
    将胚胎在4°C下在0.01%阿尔新蓝中孵育过夜，在黑暗中溶解在60mM MgCl 2 和70%乙醇中（见注5和6）。

  7. 7.

    After staining, the embryos are washed twice in 5 min, respectively, with 80% ethanol/10 mM MgCl2, 50% ethanol/10 mM MgCl2, and 25% ethanol.
    染色后，胚胎分别用80%乙醇/ 10mM 2 MgCl，50%乙醇/ 10mM MgCl 2 和25%乙醇在5分钟内洗涤两次。

  8. 8.

    Embryos are bleached again for 15 min with 3% H2O2 and 0.5% KOH to reduce the staining of soft tissues. You can see the steps 4–8 in Fig. 4.
    胚胎再次用3%H 2 O 2 和0.5%KOH漂白15分钟，以减少软组织的染色。您可以在图 4 中看到步骤 4-8。

  9. 9.

    Wash with PBS for 5 min.
    用PBS洗涤5分钟。

  10. 10.

    They are washed twice for 5 min with 25% glycerol and 0.1% KOH.
    用25%甘油和0.1%KOH洗涤两次，每次5分钟。

  11. 11.

    Stained embryos are enclosed in 50% glycerol with 0.1% KOH (see Note 7).
    染色的胚胎被封闭在50%甘油和0.1%KOH中（见注7）。

  12. 12.

    Lateral, dorsal, and ventral views were obtained under the SteREO V8/Zeiss microscope. You can see the last steps 9–12 in Fig. 5.
    在SteREO V8/蔡司显微镜下获得侧视图、背侧视图和腹视图。您可以在图 5 中看到最后步骤 9-12。

Fig. 3 图3
A schematic illustration of steps 1 to 4 of the protocol. The steps are embryos washed in an embryo medium, anesthetize the embryos, wash with P B S, and bleach the embryo.

Protocol steps 1–4 协议步骤 1–4

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Fig. 4 图4
A schematic illustration of the steps 5 to 8 of the protocol. The steps are to wash the embryo with P B S, incubate the embryo, wash the embryo after staining, and bleach the embryo.

Protocol steps 5–8 协议步骤 5–8

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Fig. 5 图5
A schematic illustration of steps 9 to 12 of the protocol. The steps are embryos washed with P B S, wash twice with glycerol and K O H, and stained embryos are enclosed in 50% glycerol and 0.1% K O H.

Protocol steps 9–12 协议步骤 9–12

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Alcian blue stained chondrocranial structures are given in Fig. 6.
阿尔新蓝色染色的软骨颅骨结构如图6所示。

Fig. 6 图6
3 Microscopic images of a stained embryo depict the chondrocranial structures, control group, D M S O exposed embryo, and propylparaben exposed embryo in A, B, C, and D, respectively

Sample images of alcian blue stained embryos at 72 hpf; (a) Chondrocranial structures, (b) control group, (c) DMSO exposed embryo, (d) propylparaben exposed embryo
72 hpf 的阿尔新蓝色染色胚胎的样本图像;（a）软骨颅结构，（b）对照组，（c）DMSO暴露胚胎，（d）对羟基苯甲酸丙酯暴露胚胎

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4 Notes 4 注意事项

  1. 1.

    Since the substance we have is a hydrated component, the molecular weight of the water is subtracted when calculating the molarity. In addition to the molecular weight of hydrated magnesium chloride MgCl2, it also contains the molecular weight of H2O. Since we have magnesium chloride MgCl2.6H2O, the molecular weight of H2O should also be considered in the calculation and subtracted when calculating moles. Otherwise, the solution will need to be more accurate. Therefore, for all solutions to be prepared, it is beneficial to control the molecular weight between the value in the protocol and the material we have.
    由于我们拥有的物质是水合成分，因此在计算摩尔浓度时会减去水的分子量。除水合氯化镁MgCl的分子量外 2 ，还含有H 2 O的分子量。由于我们有氯化镁 2 MgCl.6H 2 O，因此在计算时也应考虑H 2 O的分子量，并在计算摩尔数时减去。否则，解决方案将需要更准确。因此，对于要制备的所有溶液，控制协议中的值与我们拥有的材料之间的分子量是有益的。

  2. 2.

    According to the 2020 American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals, formaldehyde poses serious health concerns to staff members, including the potential for respiratory, dermal, and ocular irritation and hypersensitivity, along with being cancer-causing. When working with formaldehyde, it is essential to use a lab coat, mask, and gloves to avoid inhalation of the chemical and to reduce contact. While wearing gloves, it is necessary to avoid direct skin contact, such as the face and eyes.
    根据 2020 年美国兽医协会 （AVMA） 动物安乐死指南，甲醛会给工作人员带来严重的健康问题，包括潜在的呼吸道、皮肤和眼部刺激和超敏反应，以及致癌。使用甲醛时，必须使用实验室外套、口罩和手套，以避免吸入化学物质并减少接触。戴手套时，必须避免直接接触皮肤，例如面部和眼睛。

  3. 3.

    According to the guideline, to anesthetize, we can prepare a 10 g/L buffered stock solution with sodium bicarbonate with a pH of 7.0–7.5. Tricaine-S and sodium bicarbonate are mixed in a ratio of 1–2, and the stock solution is completed with distilled water according to its volume. When working with chemicals that pose a risk for retinal toxicity, eye contact should be avoided, and protective clothing should be worn. It should be stored at 4 °C and protected from light. It can be kept at −20 °C longer. Stock solution can be used for up to 6 months, and 10% solution can be kept at room temperature for 3 days [10, 11].
    根据指南，为了麻醉，我们可以制备 pH 值为 7.0-7.5 的 10 g/L 碳酸氢钠缓冲储备溶液。将Tricaine-S和碳酸氢钠以1-2的比例混合，并根据其体积用蒸馏水完成储备溶液。使用具有视网膜毒性风险的化学品时，应避免眼睛接触，并应穿防护服。应储存在4°C并避光。它可以在-20°C下保持更长时间。储备溶液可使用长达6个月，10%溶液可在室温下保存3天[10,11]。

  4. 4.

    The images of zebrafish larvae and embryos in the figures were taken from SciDraw and created via BioRender [14, 15].
    图中斑马鱼幼虫和胚胎的图像取自SciDraw，并通过BioRender创建[14,15]。

  5. 5.

    It is essential to stay within the times indicated in the protocol steps. At the end of all steps, attention should be paid to the duration to obtain the ideal image.
    必须保持在协议步骤中指示的时间内。在所有步骤结束时，应注意获得理想图像的持续时间。

  6. 6.

    Standardizing the waiting times of embryos kept for overnight staining is essential. It is necessary to stay in the same duration every time the protocol is applied and to apply the steps by keeping the time loss to a minimum.
    标准化用于过夜染色的胚胎的等待时间至关重要。每次应用协议时，都必须保持相同的持续时间，并通过将时间损失保持在最低限度来应用这些步骤。

  7. 7.

    In a 50% glycerol 0.1 KOH solution, it can be challenging to change the placement of the embryos and keep them stable. For this, positioning the embryos one by one on 70% glycerol in a separate petri dish and adjusting the light with the correct and standard angle will bring better results. It is easier to fix and image the larvae in pure glycerol or close ratios [13].
    在50%甘油0.1 KOH溶液中，改变胚胎的位置并保持它们稳定可能具有挑战性。为此，将胚胎一个接一个地放在单独的培养皿中，放在70%的甘油上，并以正确和标准的角度调整光线将带来更好的结果。在纯甘油或接近比例下对幼虫进行固定和成像更容易[13]。