需要JoVE订阅才能观看此内容。 请登录或开始免费试用

方法文章

优化的小鼠胚胎整体蛋白与转录表达发育研究方法

167 次观看

⸱

2026年9月11日

 ,  ,  ,  , 
1Department of Physiology, Anatomy and Genetics, University of Oxford, 2Cellular Imaging Core Facility, Centre for Human Genetics, Nuffield Department of Medicine, University of Oxford

本文内容

摘要

小鼠模型在推动我们对胚胎发育的理解方面发挥着关键作用。本文介绍了一种对完整固定的小鼠胚胎进行蛋白质和转录本染色的实验方案，以及如何在三维空间中对染色结果进行定量分析。这些技术可实现对关键发育细胞类型和发育过程的可视化与分析。

摘要

小鼠模型在推动我们对胚胎发育的理解方面发挥了重要作用。分析胚胎内蛋白质和转录本的表达与定位，对于阐明发育和疾病相关的细胞与分子机制至关重要。然而，传统的小鼠胚胎转录本和蛋白质分析技术存在局限性。RNA 原位 杂交可实现转录本的整体染色，但缺乏多重检测能力。相比之下，胚胎切片技术虽可实现多重靶标染色，但仅限于二维（2D）层面。近年来，RNAscope 和免疫荧光染色等技术已被应用于整体胚胎，可在三维（3D）空间中实现多重靶标检测。这些方法能够在克服传统技术局限性的同时，对胚胎发育所必需的关键相互作用、信号通路和细胞类型进行定位与描绘。

本文介绍了使用RNAscope或免疫荧光法对固定后的胚胎第8.5天（E）8.5和E9.5小鼠胚胎进行整体染色的优化方法。两种方法均可在同一胚胎中同时实现最多4个靶标的多重检测。我们还提供了一种在同一胚胎中联合使用免疫荧光染色与RNAscope的方法，以可视化发育过程中蛋白质与转录本之间的相互作用。此外，我们描述了利用蔡司Arivis Pro软件对整个胚胎以及特定发育细胞类型中靶标表达进行共聚焦成像和三维分析的工作流程。综上所述，这些方法可实现对三维空间中多重蛋白质和转录本靶标的全面分析，为研究早期至中期胚胎发生过程中的细胞与分子相互作用提供了强有力的系统。

引言

小鼠模型在推动发育生物学研究进展方面发挥了重要作用，因其允许在体内（in vivo）研究与人类相关的发育过程，这得益于小鼠与人类在发育上的同源性，并可通过Cre-LoxP或CRISPR/Cas9等关键技术调控基因表达1,2。小鼠与人类之间约90%的基因组区域是保守的3，且两者共享许多发育过程。例如，小鼠和人类的胚胎发生均遵循相似的时间进程，包括受精卵形成、发育为胚泡，随后发生植入和原肠胚形成4。此外，大多数器官的发育在小鼠和人类之间也十分相似。例如，人类和小鼠心脏均经历相似的心脏环化、心房与心室的分隔、瓣膜形成以及流出道形成等过程，最终发育为四腔心脏5。因此，发育中及完全成熟的小鼠与人类心脏在结构上具有显著的相似性6,7。

为了研究正常和病理发育过程中发生的过程，已开发出多种技术，能够实现关键蛋白质和转录本的空间可视化。这些技术包括免疫荧光染色和RNA 原位杂交。

RNA 原位 杂交技术最早由 Gall 和 Pardue8 以及 John、Brinstall 和 Jones 于 1969 年9，以及 Buongiorno-Nardelli 和 Amaldi 于 1970 年10 首次描述。该技术用于检测转录本在组织中的表达8。简而言之，选择含有与目标 RNA 互补序列的探针，并用可检测的标记物进行标记。此类标记物包括放射性标记、生物素或地高辛。固定后的样品需进行通透处理，以允许探针渗透，随后探针与组织中的靶序列杂交。若使用放射性标记，可直接对组织成像；否则可通过与碱性磷酸酶或荧光染料偶联的抗体来可视化标记信号。RNA 原位 杂交的优势在于可用于整体样品染色，实现三维定位11。然而，该技术通常受限于多重检测能力，一般只能同时检测不超过两个目标，除非采用具有特定应用的优化技术，或使用 DNA 杂交方法（如用于染色体荧光标记的 M-FISH 技术）12,13。

免疫荧光技术由 Coons 等人于 1941 年首次开发14,15，是一种用于检测固定或冷冻样本中目标蛋白的方法。简而言之，样本 需先进行封闭处理，以减少抗体与组织非靶区域的非特异性结合。随后，将针对目标抗原的特异性一抗与样本孵育。一抗可直接与荧光染料或荧光蛋白偶联，也可通过使用荧光标记的二抗进行间接检测，该二抗特异性识别一抗所来源的物种。相比直接偶联荧光基团的一抗，使用二抗的优势在于可实现信号放大，从而提高检测灵敏度。通过使用不同物种来源的一抗，并结合直接偶联或与不同激发和发射光谱的荧光染料偶联的二抗，可实现多靶标同时检测。

传统的技术如RNA原位杂交和免疫荧光存在局限性。通常，RNA原位杂交仅允许在样品中检测单一靶标，因此不同细胞类型和通路之间的相互作用研究极为受限。此外，免疫荧光传统上是在组织切片中进行，而非整体封片，导致空间分辨率局限于二维。近年来的技术发展则聚焦于在三维系统中实现多重靶标检测。免疫荧光方案的优化使其可用于整体封片样品16。此外，已开发出可通过荧光标记探针实现转录本多重检测的技术，其原理与免疫荧光类似。

RNAscope 由 Wang 等人于 2012 年首次描述，其基于 RNA 原位 杂交技术17，可在固定样本中实现转录本的空间定位。简而言之，固定后的样本经过通透处理，使靶标探针能够渗入并与互补的转录序列结合。RNAscope 探针采用“双 Z 设计”，包含一个特异性识别靶标 RNA 中 18–25 个碱基对区域的片段、一个含 14 个碱基对的尾部区域，以及连接两个“Z 探针”的间隔区域。两个尾部区域共同形成一个 28 bp 的区域，可被预扩增子结合。结合后的 Z 探针对通过预扩增子上的结合位点依次结合一系列扩增子而实现扩增。扩增子可被带有荧光标记或辣根过氧化物酶（HRP）标记的探针特异性识别。探针的双 Z 设计旨在防止未结合探针的扩增，从而减少非特异性扩增；而使用多个扩增子则可提高检测灵敏度。与传统 RNA 原位 杂交相比，RNAscope 的优势在于可通过为不同靶标使用不同的荧光标记，在单个样本中实现多重转录本检测。RNAscope 已广泛应用于福尔马林固定石蜡包埋的组织切片，可在二维空间内实现转录本的定位检测18。近期研究进一步报道了在小鼠胚胎中进行整体装片 RNAscope 染色，从而实现对关键发育过程的三维分析19。

本文介绍了对固定的小鼠E8.5和E9.5胚胎进行整体染色的优化方法，结合使用RNAscope 原位 杂交和免疫荧光技术。本方案描述了一种改进的免疫荧光与RNAscope联合染色方法，适用于整体E8.5/E9.5小鼠胚胎，可通过引入第四种RNAscope探针并结合Opal 780荧光染料，实现在整体RNAscope染色中增加一个额外的检测通道，并详细阐述了一种广泛适用的染色胚胎分析方法。这些方法可用于深入研究细胞迁移和信号通路等关键发育过程，通过免疫荧光染色和RNAscope技术在整体E8.5/E9.5小鼠胚胎中实现最多4种蛋白和转录本目标的多重可视化，并进行发育过程中的定量三维分析。

访问受限。请登录或开始试用以查看此内容。

方案

All animal experiments were performed in compliance with the UK Animals (Scientific Procedures) Act 1986 and approved by the University of Oxford animal welfare review board and the Home Office (Project license PP7967396).

1. Dissection, dehydration, and storage of E8.5 and E9.5 mouse embryos

  1. Cull pregnant females using an approved method at E8.5 or E9.5 gestation.
    NOTE: Mice in this study were culled using cervical dislocation followed by confirmation of death by exsanguination. Exsanguination is performed by making an incision in the ventricles, facilitating bleeding. Other methods, such as CO2 inhalation, may be used, but were not tested in this protocol.
  2. Open the abdomen first by cutting through the skin, then the abdominal wall.
    NOTE: Swab mice with 70% ethanol to reduce dander and bacterial contamination. When opening the abdominal wall, it is important to pull the wall away from the abdomen, then make a small incision. Cut the abdominal wall in a V-shape, avoiding inadvertently cutting the uterus.
  3. Remove the uterus by cutting where the uterus meets the vagina. For both uterine horns, cut the uterus away from the abdominal fat, making small excisions until you reach the ovary.
    NOTE: It is worthwhile to make a note of the position of each embryo within the uterus. Dissect out the ovary with the uterus to help maintain orientation.
  4. Individually cut a single embryo from the uterine horn, within the surrounding uterus, and place it in a dish containing 1× Hanks’ Basic Salt Solution with 5mM EDTA.
  5. Using fine forceps, gently peel away the uterus from the embryo, placenta, and the decidua surrounding the two.
  6. Identify where the placenta meets the part of the maternal decidua surrounding the embryo; there should be a slight difference in color between these two sections. Carefully separate the embryo by cutting through the maternal decidua to disconnect the placenta and reveal the embryo.
  7. Gently tease the embryo within its yolk sac from the remaining tissue. Using fine forceps, remove the yolk sac surrounding the embryo. Remove the amnion. Count somites to determine the exact embryonic stage.
    NOTE: The yolk sac surrounds the embryo and is thicker and more opaque. The amnion is thin, transparent, and clings tightly to the embryo. This needs to be carefully removed to ensure downstream probe/antibody penetration. For older embryos, a hole can be poked in the head with fine forceps to prevent probe trapping in the developing brain ventricles. It is important at this point to remove as much of the yolk sac as possible. Leftover yolk sac fluoresces strongly when anti-mouse secondary antibodies are used.
  8. Optional: Store the yolk sac for genotyping by washing in 500 µL of PBS or 70% ethanol, depending on downstream genotyping, in a 1.5 mL Eppendorf tube to remove contaminating blood. Hold the yolk sac in the PBS using fine forceps and manually agitate. Take the washed yolk sac and place it in a clean 1.5 mL Eppendorf tube or genotyping plate. Store for up to 4 weeks at -20 °C or proceed to the preferred DNA extraction technique.
  9. Using a 2 mL low-density polyethylene (LDPE) Pasteur pipette, gently pipette the embryo and move to a 2 mL round-bottomed Eppendorf containing 1 mL of freshly made or frozen stock 4% paraformaldehyde (PFA), taking care to add minimal dissection media to the PFA.
    NOTE: When dissecting E9.5 embryos, cut the end of the Pasteur pipette at an angle to increase the size of the opening. This is important to avoid damage to the embryo when pipetting. Use of round-bottomed 2 mL Eppendorf tubes is important to prevent the embryo from getting stuck in the bottom of the tube and allowing easier recovery. Avoid repeated freeze-thaw cycles of PFA.
    CAUTION: PFA is toxic and carcinogenic. Use appropriate personal protective equipment (PPE) and do not inhale or ingest. Powdered PFA is dangerous if inhaled; therefore, it must be prepared in a fume hood.
  10. Incubate the embryo in 4% PFA for 16–24 h at 4 °C with gentle rocking, with tubes at right angles to the direction of rocking.
  11. Dehydrate the embryo using washes in a methanol series. Using LDPE Pasteur pipettes and taking care not to pipette up the embryo, take off PFA and add 1 mL PBS. Wash for 10 min at room temperature (RT) with gentle rocking, with tubes at 90° to the plane of rocking. Repeat for washes in 25%, 50% 75% and 2× 100% methanol in PBS.
  12. Replace the last 100% methanol wash with 1.5 mL of fresh 100% methanol and store at -20 °C.
    NOTE: Samples can be stored for years at -20 °C. Take care to avoid evaporation.
    CAUTION: Methanol is flammable and toxic. Wear appropriate PPE and store in a flammables cabinet. Do not inhale or ingest.

2. Staining whole-mount E8.5 and E9.5 embryos for proteins using immunofluorescence

NOTE: All wash steps are 1 mL volume performed in a 2 mL round-bottomed Eppendorf tube. Perform 1mL wash steps with gentle rocking and tubes at 90° to the plane of rocking. Remove and replace liquid using a 2 mL LDPE Pasteur pipette, taking care not to pipette up the embryo. When using E9.5 embryos, cut the end of the Pasteur pipette to prevent damage to the embryo in the case of inadvertent pipetting. Dilute methanol in PBS. Embryos can be stained individually or in a tube with up to 12 pooled embryos. For E9.5 embryos: up to 2 conditions can be stained in a single tube. If using multiple conditions, cut the tail of one of the groups so they can be distinguished when imaging.

  1. Retrieve embryos from -20 °C storage and allow them to come to room temperature (RT) at 20–26 °C for 10–20 min. Make sure embryos have similar somite numbers to ensure comparison of similar developmental stages.
  2. Bleach and permeabilize the embryos in a solution of 100% methanol: Dimethyl sulfoxide (DMSO):30% hydrogen peroxide in a 4:1:1 ratio for 2 h.
    CAUTION: Hydrogen peroxide is corrosive and irritant. It can cause serious eye damage and be harmful if swallowed. If hydrogen peroxide is in the eye, rinse with water for several minutes. Hydrogen peroxide is an aquatic hazard; dispose of it at an approved waste disposal facility. Wear appropriate PPE, including eye protection. Do not ingest.
  3. Pre-heat the heat block to 98 °C, prepare the citrate unmasking buffer by diluting 100 µL of citrate unmasking buffer in 10.7 mL of distilled water.
  4. Rehydrate the samples by rinsing in 70% methanol, then washing in 50% methanol, 25% methanol, then PBS for 10 min each at RT with rocking.
  5. Preheat citrate unmasking buffer to 98 °C using the pre-heated heat block for at least 20 minutes. Wash the embryos in citrate unmasking buffer for 3 x 10 min at RT.
  6. After RT washes, add pre-heated citrate unmasking buffer to the embryos and heat in the heat block at 98 °C for 10 min. Remove from heat and allow to cool for at least 40 min.
    NOTE: Do not use citrate unmasking buffer for antigen retrieval in E8.5 embryos, as it can lead to embryo dissociation. Embryos can become sticky when in citrate unmasking buffer, so keep the washes stationary to avoid embryo damage due to clumping.
  7. Wash the embryos for 3 × 10 min in PBS + 0.1% Triton-X at RT with rocking. Block embryos for 16–24 h in a minimum of 150 µL/tube PBS + 0.1% Triton-X + 10% normal donkey serum at 4 °C with rocking.
  8. Prepare the primary antibodies by diluting them in PBS + 0.1% Triton-X + 10% normal donkey serum in recommended concentrations 1 h before placing on embryos and keep at 4 °C.
  9. Remove the blocking solution from the embryos and add a minimum of 150 µL/tube of the diluted antibody solution. Incubate for 1–3 days at 4 °C with rocking.
    NOTE: Primary antibodies can be multiplexed as long as each antibody is raised in a different species. Use of antibodies raised in mice, and subsequent anti-mouse secondary antibodies, can lead to bright non-specific staining. Alternatively, in some instances, primary antibodies of a single species can be multiplexed, where primary antibodies are of a different isotype and combined with isotype-specific secondary antibodies.
  10. Rinse embryos in PBS + 0.1% Triton-X, wash embryos for 6 × 30 min in PBS + 0.1% Triton-X at RT.
  11. Block secondary antibody by diluting appropriate fluorescently conjugated secondary antibodies and DAPI (or alternative nuclei marker) in 0.1% Triton-X + 10% normal donkey serum in recommended concentrations 1 h before placing on embryos at 4 °C in the dark.
  12. Remove wash and add diluted secondary antibody solution to the embryos and incubate for 16–24 h at 4 °C with rocking in the dark.
    NOTE: We recommend using F(ab) fragment fluorescently conjugated secondary antibodies rather than whole antibodies. F(ab) fragments are smaller, therefore have better tissue penetration, and the lack of the Fc region helps to reduce non-specific background.
  13. Rinse embryos in PBS + 0.1% Triton-X, then wash embryos for 6 × 30 min in PBS + 0.1% Triton-X at RT with rocking in the dark. Store for up to 4 weeks in PBS + 0.1% Triton-X at 4 °C in the dark.

3. Staining whole-mount E8.5 and E9.5 embryos for transcripts using RNAscope

NOTE: All wash steps are 1 mL in volume, performed in a 2 mL Eppendorf tube. Perform 1 mL wash steps with gentle rocking and tubes at 90° to the plane of rocking. Remove and replace liquid using a 2 mL LDPE Pasteur pipette, taking care not to pipette up the embryo. When using E9.5 embryos, cut the end of the Pasteur pipette to prevent damage to the embryo in the case of inadvertent pipetting. Methanol should be diluted with PBS. Switch on the hybridization oven prior to starting at 40 °C with some damp tissue at the bottom. Embryos can be stained individually or in a tube with up to 12 pooled embryos. For E9.5 embryos: up to 2 conditions can be stained in a single tube. If using multiple conditions, cut the tail of one of the groups so they can be distinguished when imaging.

  1. Prepare the bench, pipettes, and rack by cleaning with 70% ethanol followed by an RNase decontamination solution.
  2. Rehydrate embryos through a graded methanol series diluted in PBS of 75%, 50%, 25%, PBS, and PBS + 0.1% Tween 20, for 5 min per step with gentle rocking.
  3. Permeabilize embryos by incubating in ~75 µL (3 drops) of protease III for 20 min at RT with upright rocking.
    NOTE: Permeabilization time can be optimized by embryonic stage/size. 20 min works well for E8.5/E9.5 embryos, but longer incubations might be needed to see deeper structures in E9.5 embryos. Too long permeabilization steps can lead to embryo dissociation.
  4. Wash embryos in PBS + 0.01% Tween 20 for 3 × 5 min.
  5. Prepare RNAscope probes by putting approximately 150 µL (around 6 drops) per tube of C1 probes into an Eppendorf tube. Dilute any C2–C4 probes in the C1 probe at a concentration of 1:50 and incubate upright in a hybridization oven at 40 °C for 10 min prior to placing on embryos.
    NOTE: For each set of RNAscope probes in an experiment, ‘C’ indicates the channel designation. A C1 probe is needed, as all other probes are diluted in the C1 probe. Make sure only one probe of each channel designation is used in each mix. Probe mixes can be reused at least 1× if stored at 4 °C for up to 6 weeks.
  6. Remove the last wash and add 150 µL of probe mix to each tube of embryos. Incubate in a hybridization oven upright for 16–24 h at 40 °C.
  7. Remove probe mix and wash in 0.2× Saline-Sodium Citrate (SSC) buffer + 0.1% Tween 20 for 3 × 8 min at RT.
  8. Post-fix embryos in 4% PFA at RT with gentle rocking, with tubes at 90° to the plane of rocking.
  9. Wash in 0.2× SSC buffer + 0.1% Tween 20 for 3 × 8 min at RT.
  10. Incubate samples in approximately 150 µL (6 drops)/tube of Amp1 reagent for 30 min at 40 °C in a hybridization oven, keeping tubes upright.
  11. Wash in 0.2× SSC buffer + 0.1% Tween 20 for 3 × 8 min at RT.
  12. Incubate samples in approximately 150 µL (6 drops)/tube of Amp2 reagent for 30 min at 40 °C in a hybridization oven, keeping tubes upright.
  13. Wash in 0.2× SSC buffer + 0.1% Tween 20 for 3 × 8 min at RT.
  14. Incubate samples in approximately 150 µL (6 drops)/tube of Amp3 reagent for 15 min at 40 °C in a hybridization oven, keeping tubes upright.
  15. Wash in 0.2× SSC buffer + 0.1% Tween 20 for 3 × 8 min at RT.
  16. Incubate in 150 µL (6 drops) HRP-C1 for 15 min at 40 °C upright in a hybridization oven. Wash in 0.2× SSC buffer + 0.1% Tween 20 for 3 × 8 min at RT.
  17. Incubate samples with a fluorophore diluted in TSA buffer for 30 min at 40 °C in a hybridization oven, keeping tubes upright.
    NOTE: RNAscope fluorophores can be used on different probes. TSA-vivid 570 is the brightest, then TSA-vivid 650, then TSA-vivid 520. Select fluorophores based on expected probe abundance.
  18. Wash in 0.2× SSC buffer + 0.1% Tween 20 for 3 × 8 min at RT.
  19. In the dark, incubate samples in approximately 150 µL (6 drops)/tube of HRP-blocker reagent for 15 min at 40 °C in a hybridization oven, keeping tubes upright.
  20. Wash in 0.2× SSC buffer + 0.1% Tween 20 for 3 × 8 min at RT.
    NOTE: The protocol can be paused here for 16–24 h by leaving the embryos in the last wash at RT.
  21. Repeat steps 3.16–3.20 for each of probes C2–C4 if being used, swapping HRP-C1 for HRP C2/C3/C4 as required. Use a different fluorophore for each separate probe.
    NOTE: If using C4 probes, the RNAscope 4-plex ancillary kit and the Opal 780 fluorophore are required. Opal 780 should only be used when multiplexing of 4-probes is needed and should be the last fluorophore used, as it is sensitive to degradation. When selecting fluorophores, it is important to consider the capabilities of the microscopy platform you will be using. Always check that the imaging system has appropriate excitation wavelengths, emission filtering, and detector sensitivity for all probes used. When staining with Opal 780, replace steps 3.17–3.19 with the following:
    1. Incubate samples in 150 µL/tube TSA-DIG (diluted 1:100 in TSA-buffer) for 30 min at RT with gentle rocking.
    2. Wash in 0.2× SSC buffer + 0.1% Tween 20 for 3 × 8 min at RT.
    3. In the dark, incubate samples in approximately 150 µL (6 drops)/tube of HRP-blocker reagent for 15 min at 40° in a hybridization oven, keeping tubes upright.
    4. Wash in 0.2× SSC buffer + 0.1% Tween 20 for 3 × 8 min at RT.
    5. Incubate samples in 150 µL/tube of Opal Polaris 780 diluted in Antibody Diluent/Block for 45 min at 40 °C in a hybridization oven, keeping tubes upright
  22. In the dark, incubate samples in approximately 150 µL of DAPI diluted in 0.2 x SSC buffer + 0.1% Tween 20 (working concentration 1µg/mL) for 16–24 h at 4 °C with gentle rocking, keeping tubes upright
  23. Wash in 0.2× SSC buffer + 0.1% Tween 20 for 3 × 8 min at RT. Store samples short-term in PBS at 4 °C.

4. Combined RNAscope and immunofluorescence staining in whole-mount E8.5 embryos

  1. Follow steps 3.1–3.21 of the RNAscope protocol.
    NOTE: Reduction of the time of the RNAscope permeabilization step might help with improved antibody performance on immunofluorescence.
  2. Wash samples for 16–24 h in 1 mL of 0.2× SSC buffer + 0.1% Tween 20 at RT with rocking.
  3. Follow steps 2.7–2.12 of the immunofluorescence protocol.

5. Preparing whole-mount embryos for imaging using point scanning or spinning disk confocal microscopy

  1. After staining, using an LDPE Pasteur pipette, gently pipette up the embryo and pipette the embryo into either a multi-well imaging slide or a cavity slide, taking care to make sure the embryo does not dry out. If needed, orient the embryo as desired, taking care not to damage the sample.
    NOTE: Cavity slides are beneficial for ensuring the orientation of embryos but can lead to embryo compression and tissue damage. Multi-well imaging slides are gentle on embryos but can lead to variation in imaging orientation and may introduce edge-of-well artifacts.
  2. Optional – Tissue clearing: clear tissues by gently removing the last wash from the embryo and adding up to 200 µL of CUBIC-R solution within the imaging dish at RT. Keep the embryo in a single incubation CUBIC-R for a minimum of 40 min before imaging. Cleared samples should be imaged in the same CUBIC-R solution used for the incubation.
    CAUTION: CUBIC-R is toxic when inhaled and can lead to eye and skin irritation. Avoid inhalation and use appropriate PPE when handling.
    NOTE: Embryos become completely transparent shortly after exposure to CUBIC-R. Only add CUBIC-R directly to the imaging dish after orienting and take care not to lose the embryo. Partial reversal of clearing occurs if CUBIC-R is removed from the embryo and replaced with PBS.
  3. If not performing tissue clearing, top up wells with PBS to ensure embryos do not become too dry. If using a cavity slide, cover the slide using a #1.5 coverslip. Add a lid to the multi-chamber imaging slide.
    NOTE: Embryos can be imaged with a wide range of light microscopes. Due to sample thickness, it is pertinent to select a microscope that can image at an appropriate depth. Choose an objective appropriate for the desired structure. A 10x, low numerical aperture objective gives a good overview of whole embryo staining, but a higher magnification and numerical aperture objective might be necessary to resolve smaller or deeper structures in high resolution. Embryos can sometimes move in the imaging dish, introducing errors, so reducing imaging time where possible is important.

6. Analyzing nuclei for counts and signal intensity

NOTE: This protocol covers analysis of nuclei using Zeiss Arivis Pro; similar analyses may be possible using alternative image analysis programs. It works best with samples that are nuclear, e.g., transcripts or nuclear proteins. If analyzing non-nuclear proteins, it may be worth using a stain to visualize cell structure, e.g., CellMask, which stains cell membranes, and examining the expression of markers within the entire cell.

  1. Convert imaging files into SIS format using Zeiss Arivis SIS converter. Open the converter and select ‘Add files,’ then ‘Browse folder’. Select the source image folder. Select 1-to-1 conversion. Choose your output directory and select ‘start conversion’.
  2. Import SIS files into Zeiss Arivis Pro by selecting ‘file,’ then ‘open,’ then select the desired SIS file.
  3. Prepare the data for pipeline analysis by rotating images. To do so, select ‘Data’ then ‘Transformation gallery’. Select the ‘rotation’ function and rotate the embryo so the region of interest can align into a rigid rectangular box.
  4. Crop regions of interest by selecting ‘Data,’ then ‘Transformation gallery,’ selecting the ‘Crop’ option. Drag edges to crop to a region of interest and select ‘Ok’.
    NOTE: For the head region in E8.5 embryos, crop region to head, cutting off the image at the notch close to the otic vesicle. For the heart region of E8.5 embryos, crop from below the notch close to the otic vesicle, to the bottom of the heart tube. When cropping rotated images, crop out the black regions created as an artifact of the rotation.
  5. Start a new pipeline by selecting ‘Analysis’ then ‘Analysis Panel’. Select ‘new pipeline’.
  6. Input ROI: Set ROI to ‘current image set’, set channels to ‘All channels,’ and scaling to ‘100%’. Ensure ‘include Z’ is selected.
    NOTE: Images can be downscaled (e.g., to 50%) if they are too large or processing is too slow; this will reduce the resolution of the images.
  7. Find nuclei (marker #1) using the blob finder tool: Select ‘Add Operation,’ then find ‘Blob finder’. In the channel, select the channel containing the nuclei marker. Set the diameter to 6 µm and optimize the probability threshold based on staining intensity. Nuclei in the plane should be detected with appropriate separation between the nuclei. Set the split sensitivity. 55% is a reasonable value for most samples. Set ‘Normalization’ to the first time-point. Set output name, e.g., Blob finder (nuclei). Detected objects can be visualized by selecting the ‘eye’ icon in the top bar of the operation.
  8. Find the expression of marker #2 using the blob finder tool: Select ‘Add Operation’, then find ‘Blob finder’. In the channel, select the channel containing the nuclei marker. Set the diameter to 6 µm and optimize the probability threshold based on staining intensity. ‘Blobs’ positive for marker #2 in the plane should be detected with appropriate separation. Set the split sensitivity. 55% is a reasonable value for most samples. Set ‘Normalization’ to the first time-point. Set output name, e.g., Blob finder (channel 2). Detected objects can be visualized by selecting the ‘eye’ icon in the top bar of the operation.
  9. Find the expression of marker #3 using the blob finder tool: Select ‘Add Operation,’ then find ‘Blob finder’. In the channel, select the channel containing the nuclei marker. Set the diameter to 6 µm and optimize the probability threshold based on staining intensity. ‘Blobs’ positive for marker #3 in the plane should be detected with appropriate separation. Set the split sensitivity. 55% is a reasonable value for most samples. Set ‘Normalization’ to µ first time-point. Set output name, e.g., Blob finder (channel 3). Detected objects can be visualized by selecting the ‘eye’ icon in the top bar of the operation.
  10. Optional: volume filter – If there is a lot of debris in imaging, a volume filter can be used to remove artifacts. Select ‘Add Operation,’ then find ‘Object Feature Filter’. Set input to Blob finder (nuclei) (or other set name from step 6.7). Set ‘is type’ to ‘any’. In the dropdown menu, select Volume (volume) and set the value to > 6 µm3. Select ‘Add filter’. In the dropdown menu, select Volume (volume) and set the value to <3000 µm3. Name the output, e.g., ‘nuclei’. Run the pipeline to here by selecting the 3 lines button in the module and clicking ‘run pipeline to here’.
    NOTE: Volume filters can also be used for the other channels if necessary. Optimize the greater than and less than values to ensure all positive objects are being selected, but omitting debris.
  11. To identify nuclei that are positive for one other marker, use the intersect tool. Select ‘Add operation’ and find the ‘compartments’ tool. In the first dropdown box, add the nuclei output (e.g., blob finder (nuclei) or the volume filtered ‘nuclei’) and ensure the button to the left of the box is selected. In the inset dropdown box, select the desired channel (e.g., blob finder (channel 2)). Name the output, e.g., ‘nuclei with channel 2’. Repeat for all desired markers.
  12. To find nuclei that are positive for 2 or more other markers, select ‘Add operation’ and find the ‘Object Feature Filter’ tool. Set the input to the nuclei marker (e.g., ‘nuclei’, type= any and add filters from the previous step after ‘Has tag,’ e.g. Nuclei with channel 2’ and ‘Nuclei with channel 3’. Name output, e.g., ‘Nuclei with channel 2 and channel 3’. Run the pipeline to here
  13. Create custom features to count nuclei by selecting the data table icon in the top bar, selecting ‘create,’ and selecting ‘object count’. Give the feature an appropriate label (e.g., nuclei count) and select the desired output to quantify (e.g., nuclei) in the tag menu. Repeat this step for each group to be quantified.
  14. Create object groups by selecting ‘Add operation’ then ‘Create object groups’. Select each of the groups to be used in the analyses in the inputs. Label outputs as ‘Create object groups’. Run pipeline to here.
  15. Export summary counts by selecting ‘Add operation’ then ‘Export object features’. In inputs, select ‘create object groups’. Select a directory to save data. Mode is ‘single table’. Click features, and select the custom features created in step 6.13.
  16. Export nuclei detail by selecting ‘Add operation’ then ‘Export object features’. In inputs, select the nuclei detection created in step 6.7 (or 6.10 if using a volume filter). Select a directory to save data. Mode is ‘single table’. Click features, and select ‘intensities’ for each channel (#1, #2, #3, etc.) and other desired features to export, e.g., volume (volume).
  17. Store objects by selecting the objects needed for visualization/analysis in the store objects box.
  18. Save pipeline by reverting all steps, selecting the 3 lines in the top right, and clicking ‘Export’. Select ‘folder’ to save ‘pipeline’ in and save.
  19. To batch run a pipeline, reload Arivis. In the top bar, select ‘Analysis’ then ‘Batch analysis’. In the window, select the pipeline saved in step 6.18. In ‘files’ select the files for analysis and select ok. Once loaded in, any unwanted views can be deleted. Select ‘Ok’. In the next screen, select the directory to save data. Click ‘Run’. Data will automatically save in the selected output folder.

An overview of the workflow is shown in Figure 1.

Embryo harvest and wholemount immunofluorescence and RNAscope diagram; confocal microscopy analysis.
Figure 1: Immunofluorescence and RNAscope staining protocol. Schematic representing key stages of embryo harvest, immunofluorescence, RNAscope, combined immunofluorescence and RNAscope, sample imaging, and analysis. Colored boxes correspond to different protocol stages. Created in BioRender. Sparrow, D. (2026) https://BioRender.com/q6rmax8 Please click here to view a larger version of this figure.

访问受限。请登录或开始试用以查看此内容。

结果

采用免疫荧光染色法对E8.5胚胎进行染色，以分析处于细胞周期G2/M期的迁移性神经嵴细胞数量。样品使用靶向PHH3的抗体染色以显示细胞周期的G2/M期，同时使用SOX10抗体标记迁移性神经嵴细胞。随后使用相应的二抗进行染色。细胞核用DAPI染色。使用Olympus SpinSR转盘共聚焦显微镜配备10倍物镜（NA = 0.4）对样品成像。图像通过Hamamatsu Orca Fusion BT相机和50 µm针孔转盘采集，并采用平铺拼接功能以Z栈形式获取，Z轴间距为1.99 µm。神经嵴细胞通过上皮-间质转化（EMT）实现脱离及后续迁移，此过程要求迁移前的神经嵴细胞处于细胞周期的S期或G2/M期20。在此，我们可观察到在迁移性神经嵴细胞群体中处于细胞周期G2/M期的细胞，其由PHH3染色（绿色）指示，而迁移性神经嵴细胞由SOX10染色（品红色）指示（图2A）。可通过定量对照组与处理组胚胎中处于G2/M期的迁移性神经嵴细胞数量，判断处理是否影响了处于G2/M期的迁移性神经嵴细胞数量，从而推断该处理是否也可能干扰通过EMT介...

访问受限。请登录或开始试用以查看此内容。

讨论

本文介绍了一种优化的方法，用于在E8.5和E9.5全胚胎标本中空间分析基因和蛋白质的表达。与传统的RNA 原位 杂交和组织切片免疫荧光方法相比，该方法具有更高的空间分辨率和多重检测能力。本方案在现有最新方案的基础上，进一步用于研究小鼠胚胎的发育通路。最近一项研究利用RNAscope与免疫荧光染色技术检测胚胎中的Wnt信号通路，但其染色对象为组织切片而非全胚胎标本26。另一项近期研究对E8.5至E10.5阶段的全胚胎小鼠标本进行了全胚胎RNAscope和免疫荧光染色27，但作者未将两种染色技术结合使用，也未使用C4探针和Opal 780荧光染料实现第四通道的RNAscope染色。联合使用免疫荧光与RNAscope技术此前已在E3.5全胚胎中有所报道，其中RNAscope在完成免疫荧光流程后进行28。尽管我们尚未在较晚期胚胎中尝试该确切方案，但在E8.5胚胎中，若将RNAscope染色夹在免疫荧光的一抗与二抗孵育之间，则抗体染色效果较差。这可能是由于对较大胚胎进行RNAscope时...

访问受限。请登录或开始试用以查看此内容。

披露

作者声明无竞争性财务利益。

致谢

作者感谢牛津大学人类遗传学中心细胞成像核心设施团队为本手稿中涉及的显微镜技术和图像分析提供的技术支持。作者感谢牛津大学生物医学服务中心工作人员在动物饲养以及定时交配以获取胚胎方面给予的支持。

本研究的资助来自英国心脏基金会高级研究员项目 FS/SBSRF/22/31022（DBS）、Additional Ventures 单心室基金（DBS）、牛津大学 John Fell 基金 0016065（VSR）以及牛津大学医学科学内部基金 0016707（VSR）。

访问受限。请登录或开始试用以查看此内容。

材料

本文使用的材料清单
姓名公司目录编号评论
塑料器皿/玻璃器皿
3 ml 胎牛血清移液管，低密度聚乙烯（LDPE）带刻度Wheaton711116
Eppendorf 安全锁 1.5 ml 微量离心管Eppendorf0030 120.086
Ibidi µ-slide 18 孔板 Ibidi81817#1.5H 玻璃盖玻片，未处理
Menzel X1000 盖玻片 22×50 mm #1.5 (0.16–0.19 mm)Thermo Scientific17284904
带凹槽的载玻片Marienfeld Superior1320000
安全密封微量离心管 2 mlSarstedt72.695.5002 ml “Eppendorf”管，圆底
溶液/缓冲液
1× 抗体稀释/封闭液Akoya BiosciencesARD1001EA
Ambion™ RnaseZap™ RNase 去污染溶液Ambion9780.9782
柠檬酸盐抗原修复缓冲液Vector labs H-3300标记为抗原修复液，
柠檬酸为基础
二甲基亚砜（DMSO）Merck (Sigma-Aldrich)D8418
Hanks' 平衡盐溶液 10×Merck (Sigma-Aldrich)H4641
过氧化氢溶液Merck (Sigma-Aldrich)H100930% (w/w) 水溶液
甲醇Merck (Sigma-Aldrich)32213-2.5L-M
正常驴血清 AbcamAb7475100% 储存浓度，
最终浓度 10%
多聚甲醛（PFA）粉末Merck (Sigma-Aldrich)158127-500g用水稀释至 4%。在通风橱中配制。加热搅拌，并加入少量 NaOH 以促进溶解。pH 7.2–7.6
磷酸盐缓冲液（PBS）Merck (Sigma-Aldrich)P4417每 200 ml 蒸馏水溶解 1 片
RNAscope 4-Plex 辅助试剂盒（用于多重荧光检测试剂盒 v2）ACD (Bio-Techne)323120包含 HRP-C4 和 HRP 封闭剂
RNAscope™ 多重荧光检测试剂盒 v2 ACD (Bio-Techne)323110包含蛋白酶 Plus、蛋白酶 III、
蛋白酶 IV、过氧化氢、TSA 缓冲液、
Amp1/2/3、Hrp-C1/2/3、HRP-封闭剂和 DAPI
盐水-柠檬酸钠（SSC）缓冲液 20× 溶液Fisher BioreagentsBP-1325-1用蒸馏水稀释至 0.2×
组织透明试剂 Cubic-R+(M) [用于动物]Tokyo Chemical IndustryT3741
Triton™ X-100Merck (Sigma-Aldrich)X100
Tween® 20Merck (Sigma-Aldrich)P7949
Ultrapure™ 0.5 M EDTA，pH 8Invitrogen15575020
一抗
内粘蛋白抗体（V.7C7） Santa CruzSC-65495使用 1:50 稀释。 
胰岛素瘤相关抗原 1 和 2 抗体发育研究杂交瘤库34.4DS-5用甘油稀释 50%。使用 1:50 稀释。
PCNA（PC10）小鼠单克隆抗体Cell Signaling Technology2586S使用 1:500 稀释
磷酸化组蛋白 H3（Ser10）（6G3）小鼠单克隆抗体Cell Signaling Technology9706S使用 1:500 稀释
磷酸化组蛋白 H3（Ser10）（D7N8E）XP® 兔单克隆抗体Cell Signaling Technology53348S使用 1:500 稀释
Sox10（E6B6I）XP® 兔单克隆抗体 Cell Signaling Technology69661S使用 1:500 稀释
二抗/核染料
AlexaFluor® 488 标记的 Affinipure® 驴抗小鼠 IgG（H+L）Jackson Immunoresearch715-545-151使用 1:500 稀释
AlexaFluor® 647 标记的 Affinipure®  驴抗大鼠 IgG（H+L）Jackson Immunoresearch712-605-153使用 1:500 稀释
AlexaFluor® 647 标记的 Affinipure® F(ab')2 片段驴抗小鼠 IgG（H+L）Jackson Immunoresearch715-606-150使用 1:500 稀释
Cy™3 标记的 Affinipure® 驴抗兔 IgG（H+L）Jackson Immunoresearch711-165-152使用 1:500 稀释
DAPIMerck (Sigma-Aldrich)D9542使用浓度为 1 µg/ml
RNAscope 探针
RNAscope® 探针 - Mm Sp5-C4ACD (Bio-Techne)405681-C4C4 探针
RNAscope® 探针 - Mm-Dlx2-C3ACD (Bio-Techne)555951-C3C3 探针
RNAscope® 探针 - Mm-Msx1ACD (Bio-Techne)421841C1 探针
RNAscope® 探针 - Mm-Sox10-C2ACD (Bio-Techne)435931-C2C2 探针
RNAscope 荧光染料
Opal 780 试剂包Akoya BiosciencesFP1501001KT 包含 Opal 780 试剂、Opal TSA-DIG、
DMSO
TSA-vivid™ 荧光染料 520ACD (Bio-Techne)323271现为 ClariTSA™ 荧光染料 520
TSA-vivid™ 荧光染料 570ACD (Bio-Techne)323272现为 ClariTSA™ 荧光染料 570
TSA-vivid™ 荧光染料 650ACD (Bio-Techne)323273现为 ClariTSA™ 荧光染料 650
仪器/软件
Fluoview FV4000 激光扫描  显微镜Evidentfv4000激发激光：405/488/561/640/730 nm。
检测波长（图 3）：430–470、500–540、570–620、
650–710、750–850 nm。检测波长（图 4）：430–470、
500–531、570–620 nm。
Graphpad Prism 10Graphpadv10.6.1
加热金属块GrantQBT1
Hybrigene 杂交炉Techne (Bibby Scientific)FHB4DD
Ixplore IX83 SpinSR 超分辨率显微镜系统Olympusixplore-ix83-spinsr激发激光：405/488/561/640 nm。
发射滤光片：447/60、525/50、617/73、685/40。 
Zeiss Arivis Pro Carl Zeiss Microscopy GmbHv. 4.5（原为 Arivis Vision 4D）
Zeiss Arivis SIS 转换器Carl Zeiss Microscopy GmbHv. 4.3.0

参考文献

  1. Bruter AV, Varlamova EA, Okulova YD, Tatarskiy VV, Silaeva YY, Filatov MA. Genetically modified mice as a tool for the study of human diseases. Mol Biol Rep. 2024;51(1):135.
  2. Rashbrook VS, Brash JT, Ruhrberg C. Cre toxicity in mouse models of cardiovascular physiology and disease. Nat Cardiovasc Res. 2022;1:806–16.
  3. Breschi A, Gingeras TR, Guigo R. Comparative transcriptomics in human and mouse. Nat Rev Genet. 2017;18(7):425–40.
  4. Mole MA, Weberling A, Zernicka-Goetz M. Comparative analysis of human and mouse development: From zygote to pre-gastrulation. Curr Top Dev Biol. 2020;136:113–38.
  5. Krishnan A, Samtani R, Dhanantwari P, Lee E, Yamada S, Shiota K, et al. A detailed comparison of mouse and human cardiac development. Pediatr Res. 2014;76(6):500–7.
  6. Anderson RH, Bamforth SD. Morphogenesis of the Mammalian Aortic Arch Arteries. Front Cell Dev Biol. 2022;10:892900.
  7. Wessels A, Sedmera D. Developmental anatomy of the heart: a tale of mice and man. Physiol Genomics. 2003;15(3):165–76.
  8. Gall JG, Pardue ML. Formation and detection of RNA-DNA hybrid molecules in cytological preparations. Proc Natl Acad Sci U S A. 1969;63(2):378–83.
  9. John HA, Birnstiel ML, Jones KW. RNA-DNA hybrids at the cytological level. Nature. 1969;223(5206):582–7.
  10. Buongiorno-Nardelli M, Amaldi F. Autoradiographic detection of molecular hybrids between RNA and DNA in tissue sections. Nature. 1970;225(5236):946–8.
  11. Dakou E, Vanbekbergen N, Corradi S, Kemp CR, Willems E, Leyns L. Whole-mount in situ hybridization (WISH) optimized for gene expression analysis in mouse embryos and embryoid bodies. Methods Mol Biol. 2014;1211:27–40.
  12. Anderson R. Multiplex fluorescence in situ hybridization (M-FISH). Methods Mol Biol. 2010;659:83–97.
  13. Kearney L. Multiplex-FISH (M-FISH): technique, developments and applications. Cytogenet Genome Res. 2006;114(3-4):189–98.
  14. Burnett R, Guichard Y, Barale E. Immunohistochemistry for light microscopy in safety evaluation of therapeutic agents: an overview. Toxicology. 1997;119(1):83–93.
  15. Coons AH, Creech HJ, Jones RN. Immunological properties of an antibody containing a fluorescent group. P Soc Exp Biol Med. 1941;47(2):200–2.
  16. Bardot E, Tzavaras N, Benson DL, Dubois NC. Quantitative Whole-mount Immunofluorescence Analysis of Cardiac Progenitor Populations in Mouse Embryos. J Vis Exp. 2017(128).
  17. Wang F, Flanagan J, Su N, Wang LC, Bui S, Nielson A, et al. RNAscope: a novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues. J Mol Diagn. 2012;14(1):22–9.
  18. Erben L, Buonanno A. Detection and Quantification of Multiple RNA Sequences Using Emerging Ultrasensitive Fluorescent In Situ Hybridization Techniques. Curr Protoc Neurosci. 2019;87(1):e63.
  19. De Bono C, Lescroart F, Zaffran S. How to Study Gene Expression and Gain of Function of Hoxb1 in Mouse Heart Development. Methods Mol Biol. 2025;2889:121–37.
  20. Zhao R, Moore EL, Gogol MM, Unruh JR, Yu Z, Scott AR, et al. Identification and characterization of intermediate states in mammalian neural crest cell epithelial to mesenchymal transition and delamination. Elife. 2024;13.
  21. Guijarro C, Kelly RG. On the involvement of the second heart field in congenital heart defects. C R Biol. 2024;347:9–18.
  22. Bradshaw L, Chaudhry B, Hildreth V, Webb S, Henderson DJ. Dual role for neural crest cells during outflow tract septation in the neural crest-deficient mutant Splotch(2H). J Anat. 2009;214(2):245–57.
  23. Zawada D, Kornherr J, Meier AB, Santamaria G, Dorn T, Nowak-Imialek M, et al. Retinoic acid signaling modulation guides in vitro specification of human heart field-specific progenitor pools. Nat Commun. 2023;14(1):1722.
  24. Rabadan MA, Herrera A, Fanlo L, Usieto S, Carmona-Fontaine C, Barriga EH, et al. Delamination of neural crest cells requires transient and reversible Wnt inhibition mediated by Dact1/2. Development. 2016;143(12):2194–205.
  25. Bhattacharya D, Rothstein M, Azambuja AP, Simoes-Costa M. Control of neural crest multipotency by Wnt signaling and the Lin28/let-7 axis. Elife. 2018;7.
  26. Kishimoto K, Furukawa KT, Luz-Madrigal A, Yamaoka A, Matsuoka C, Habu M, et al. Bidirectional Wnt signaling between endoderm and mesoderm confers tracheal identity in mouse and human cells. Nat Commun. 2020;11(1):4159.
  27. De Bono C, Liu Y, Ferrena A, Valentine A, Zheng D, Morrow BE. Single-cell transcriptomics uncovers a non-autonomous Tbx1-dependent genetic program controlling cardiac neural crest cell development. Nat Commun. 2023;14(1):1551.
  28. Hazra R, Spector DL. Simultaneous visualization of RNA transcripts and proteins in whole-mount mouse preimplantation embryos using single-molecule fluorescence in situ hybridization and immunofluorescence microscopy. Front Cell Dev Biol. 2022;10:986261.
  29. Kann AP, Krauss RS. Multiplexed RNAscope and immunofluorescence on whole-mount skeletal myofibers and their associated stem cells. Development. 2019;146(20).

访问受限。请登录或开始试用以查看此内容。

重印与许可

探索更多文章

整体染色蛋白表达RNAscope染色免疫荧光染色多重检测共聚焦成像三维分析胚胎发育

本文已发表

视频即将推出

联系我们
向图书馆推荐

隐私政策

使用条款

政策

版权所有 © 2026 MyJoVE Corporation。保留所有权利。