이 콘텐츠를 보려면 JoVE 구독이 필요합니다. 로그인하거나 오늘 무료 평가판을 시작하세요.

방법 논문

전체 마운트 마우스 배아의 발달 단계별 단백질 및 전사체 발현 조사를 위한 최적화된 방법

167 조회수

⸱

2026년 9월 11일

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

이 논문에서

요약

마우스 모델은 배아 발달에 대한 이해를 증진하는 데 매우 중요한 역할을 합니다. 본 영상에서는 전체 고정된 마우스 배아 내의 단백질과 전사체를 염색하는 프로토콜과, 그 결과로 나타난 염색 양상을 3차원으로 정량 분석하는 방법을 설명합니다. 이러한 기술을 통해 핵심적인 발달 세포 유형과 과정을 시각화하고 분석할 수 있습니다.

초록

마우스 모델은 배아 발달에 대한 우리의 이해를 증진시키는 데 중요한 역할을 해왔습니다. 배아 내 단백질과 전사체의 발현 및 국소화를 분석하는 것은 발달과 질병의 기저에 있는 세포 및 분자적 메커니즘을 규명하는 데 매우 중요합니다. 하지만 마우스 배아의 전사체와 단백질을 분석하는 데 사용되는 기존 기술에는 한계가 있습니다. RNA in situ 하이브리드화는 전사체의 전체 마운트 염색을 가능하게 하지만, 다중 분석 능력이 부족합니다. 반면, 배아 절편 제작은 표적의 다중 염색을 가능하게 하지만, 2차원(2D)으로만 분석할 수 있다는 제약이 있습니다. 최근에는 RNAscope 및 면역형광 염색과 같은 기술이 전체 마운트 배아에 적용되어, 3차원(3D)에서 표적의 다중 분석이 가능해졌습니다. 이러한 방법들은 기존 기술의 한계 없이 배아 발달에 필수적인 주요 상호작용, 경로 및 세포 유형의 매핑을 가능하게 합니다.

본 논문에서는 RNAscope 또는 면역형광법을 사용하여 고정된 배아기(E) 8.5 및 E9.5 마우스 배아의 전체 마운트 염색(whole-mount staining)을 위한 최적화된 방법을 설명합니다. 두 방법 모두 단일 배아에서 최대 4개의 타겟을 동시에 다중 검출할 수 있게 합니다. 또한, 단일 배아 내에서 면역형광 염색과 RNAscope를 결합하여 발달 과정 중 단백질과 전사체 사이의 상호작용을 시각화하는 방법을 제공합니다. 나아가, Zeiss Arivis Pro 소프트웨어를 사용하여 배아 전체 및 특정 발달 세포 유형 내 타겟 발현에 대한 공초점 이미징 및 3D 분석 파이프라인을 설명합니다. 이러한 방법들을 통해 3D 상에서 다중 단백질 및 전사체 타겟에 대한 종합적인 분석이 가능하며, 이는 초기 및 중기 배아 발생 과정의 세포 및 분자적 상호작용을 분석하는 강력한 시스템을 제공합니다.

서론

마우스 모델은 마우스와 인간 발달 과정 사이의 상동성과 Cre-LoxP 또는 CRISPR/Cas91,2와 같은 핵심 기술을 이용한 유전자 발현 조절 능력을 통해, 인간과 관련된 발달 과정을 in vivo에서 이해할 수 있게 함으로써 발달 생물학의 진전에 중요한 역할을 해왔습니다. 게놈 영역의 약 90%가 마우스와 인간 사이에서 보존되어 있으며3, 마우스와 인간은 많은 발달 과정을 공유합니다. 예를 들어, 마우스와 인간 모두 접합자의 형성에서 시작하여 배반포로 진행되고, 이어서 착상과 낭배 형성으로 이어지는 유사한 배아 발생 일정을 따릅니다4. 또한, 대부분의 장기 발달 과정이 마우스와 인간 사이에서 유사합니다. 예를 들어, 인간과 마우스의 심장은 모두 유사한 심장 루핑, 심방 및 심실의 중격 형성, 판막 형성, 유출로 형성 과정을 거쳐 4심실 심장을 형성합니다5. 따라서 발달 중인 심장과 완전히 발달한 심장 모두 마우스와 인간 사이에서 상당한 구조적 유사성을 보입니다6,7.

정상 및 병리적 발달 과정에서 일어나는 현상을 조사하기 위해, 주요 단백질과 전사체를 공간적으로 시각화할 수 있는 다양한 기술들이 개발되었습니다. 이러한 기술에는 면역형광 염색과 RNA in situ 하이브리드화가 포함됩니다.

RNA in situ hybridization은 1969년에 Gall과 Pardue8 및 John, Brinstall, Jones9에 의해, 그리고 1970년에 Buongiorno-Nardelli와 Amaldi10에 의해 처음 기술되었다. 이 방법은 조직 내 전사체 발현을 관찰하는 데 사용된다8. 간단히 설명하면, 원하는 표적에 상보적인 RNA 서열을 포함하는 프로브를 선택하고 검출을 위한 마커로 표지한다. 이러한 표지로는 방사성 표지, biotin 또는 digoxigenin이 있다. 고정된 시료를 투과화하여 프로브가 침투할 수 있게 한 다음, 프로브를 조직에 하이브리드화한다. 이후 조직을 직접 이미징하거나(방사성 표지의 경우), 알칼리성 포스파타아제 또는 형광 염료가 결합된 항체를 사용하여 표지를 시각화할 수 있다. RNA in situ hybridization은 3D 위치 파악을 위해 whole-mount 시료를 염색할 수 있다는 장점이 있다11. 그러나 매우 특정한 응용 분야에 최적화된 기술을 사용하거나, 염색체 형광 표지에 사용되는 M-FISH와 같은 DNA hybridization을 사용하지 않는 한, 일반적으로 2개 이상의 표적에 대한 멀티플렉싱 능력이 부족하다는 제한이 있다12,13.

1941년 Coons 등에 의해 처음 개발된 면역형광법.14,15은 고정 또는 동결된 샘플에서 표적 단백질을 검출하는 방법입니다. 간단히 설명하면, 샘플을 조직의 비표적 영역에 대한 비특이적 결합을 줄이기 위해 블로킹을 수행합니다. 그 다음, 표적 항원에 특이적인 1차 항체를 시료와 함께 배양합니다. 1차 항체는 형광 염료 또는 단백질에 직접 접합될 수 있으며, 또는 1차 항체가 생성된 종을 표적으로 하는 형광 접합 2차 항체를 사용하여 표적화할 수 있습니다. 직접 접합된 1차 항체보다 2차 항체를 사용할 때의 이점은 신호가 증폭되어 감도가 더 높아진다는 것입니다. 서로 다른 종에서 생성된 여러 1차 항체를 사용하고, 직접 접합된 항체 또는 서로 다른 들뜸 및 방출 스펙트럼을 가진 형광체에 접합된 2차 항체를 이용하면 표적 검출의 다중화(multiplexing)가 가능합니다.

RNA와 같은 전통적인 기법들은 인시튜(in situ) 하이브리다이제이션(hybridization)과 면역형광법(immunofluorescence)은 한계가 있습니다. 일반적으로 RNA 인 시투 (in situ) 하이브리드화(hybridization)는 시료 내에서 단일 표적만을 검출할 수 있으므로, 서로 다른 세포 유형 및 경로 간의 상호작용 분석에는 매우 제한적입니다. 또한, 면역형광법은 전통적으로 전체 마운트(whole-mount) 방식보다는 조직 절편에서 수행되어 공간 해상도가 2D로 제한되었습니다. 최근의 기술들은 3D 시스템 내에서 표적 검출을 다중화(multiplexing)하는 데 집중해 왔습니다. 면역형광 프로토콜의 최적화를 통해 전체 마운트 방식에서도 해당 기술을 사용할 수 있게 되었습니다.16또한, 면역형광법과 유사한 방식으로 형광 표지 프로브를 사용하여 전사체의 검출을 다중화할 수 있는 기술들이 개발되었습니다.

RNAscope는 2012년 Wang 등에 의해 처음 기술되었습니다. 이는 고정된 시료 내 전사체의 공간적 식별을 가능하게 하는 RNA in situ 하이브리드화 기술17을 기반으로 합니다. 간단히 설명하자면, 고정된 시료를 투과화하여 상보적인 전사체 서열에 결합하는 표적 프로브가 침투할 수 있도록 합니다. RNAscope 프로브는 표적 RNA의 18–25 base pair 영역에 특이적인 영역, 14 base pair를 포함하는 꼬리 영역, 그리고 두 개의 ‘Z 프로브’를 연결하는 스페이서 영역으로 구성된 ‘double-Z 디자인’을 갖추고 있습니다. 두 개의 꼬리 영역이 합쳐져 pre-amplifier가 결합하는 28bp 영역을 형성합니다. 결합된 Z 프로브 쌍은 pre-amplifier의 부위에 결합하는 일련의 amplifier 결합을 통해 증폭됩니다. Amplifier는 형광 마커 또는 horseradish peroxidase (HRP)가 표지된 프로브를 사용하여 특이적으로 표적화할 수 있습니다. 프로브의 double Z 디자인은 결합되지 않은 프로브의 증폭을 방지하여 비특이적 증폭을 줄이기 위한 것이며, 다수의 amplifier를 사용하는 것은 민감도를 높이기 위함입니다. RNA in situ 하이브리드화와 비교하여 RNAscope의 장점은 각 표적에 대해 서로 다른 형광 마커를 사용하여 단일 시료에서 전사체 검출을 다중화할 수 있다는 점입니다. RNAscope는 시료의 포르말린 고정 파라핀 임베딩 절편에 널리 사용되어 2D 상의 공간적 전사체 검출을 가능하게 했습니다18. 보다 최근의 연구들은 생쥐 배아의 whole-mount RNAscope 염색을 기술하여 주요 발생 과정의 3D 분석을 가능하게 했습니다19.

본 논문에서는 RNAscope in situ 하이브리다이제이션과 면역형광법을 모두 사용하여 고정된 E8.5 및 E9.5 마우스 배아를 전체 마운트(whole-mount)로 염색하는 최적화된 방법을 설명합니다. 이 프로토콜은 전체 마운트 E8.5/E9.5 배아에 성공적으로 적용 가능한 면역형광법과 RNAscope의 결합 개정 방법을 설명하며, Opal 780 형광체와 결합된 네 번째 RNAscope 프로브를 이용한 염색을 통합함으로써 전체 마운트 RNAscope 염색에 추가 채널을 더하는 방법을 제시하고, 염색된 배아 분석을 위한 광범위하게 적용 가능한 방법을 상세히 다룹니다. 이러한 방법들은 면역형광 염색과 RNAscope를 통해 최대 4개의 단백질 및 전사체 표적을 다중 시각화하고, 발달 중인 전체 마운트 E8.5/E9.5 마우스 배아에서 정량적 3D 분석을 가능하게 함으로써 세포 이동 및 신호 전달 경로와 같은 핵심 발달 과정에 대한 지식을 더욱 발전시키는 데 활용될 수 있습니다.

액세스가 제한되었습니다. 이 콘텐츠를 보려면 로그인하거나 체험판을 시작하세요.

프로토콜

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.

액세스가 제한되었습니다. 이 콘텐츠를 보려면 로그인하거나 체험판을 시작하세요.

결과

E8.5 embryos were stained by immunofluorescence following this protocol to analyze the number of migrating neural crest cells that were in the G2/M phase of the cell cycle. Samples were stained using antibodies targeting PHH3 to visualize the G2/M phase of the cell cycle and SOX10 to visualize migrating neural crest cells. Embryos were further stained with appropriate secondary antibodies. Nuclei were stained with DAPI. Samples were imaged using the Olympus SpinSR spinning disk confocal microscope with a 10x objective (N...

액세스가 제한되었습니다. 이 콘텐츠를 보려면 로그인하거나 체험판을 시작하세요.

토론

Here, we describe an optimized method of analyzing gene and protein expression spatially within whole-mount E8.5 and E9.5 embryos. These methods have improved spatial resolution and multiplexing ability compared to traditional methods of RNA in situ hybridization and immunofluorescence of sections. This protocol builds upon existing recent protocols to examine developmental pathways in mouse embryos. One recent protocol used RNAscope and immunofluorescence staining to look at Wnt signaling in embryos, but staine...

액세스가 제한되었습니다. 이 콘텐츠를 보려면 로그인하거나 체험판을 시작하세요.

공개 사항

저자들은 상충하는 경제적 이해관계가 없음을 밝힙니다.

감사의 글

저자들은 본 원고에 포함된 현미경 관찰 및 이미지 분석에 대한 기술적 지원을 제공해 준 옥스퍼드 대학교 인체 유전학 센터(Centre for Human Genetics)의 세포 이미징 코어(Cellular Imaging Core) 시설 팀에 감사를 표합니다. 또한, 배아 생성을 위한 동물 관리 및 적기 교배 지원을 제공한 옥스퍼드 대학교 생의학 서비스(Biomedical Services) 직원들에게 감사를 표합니다.

본 연구를 위한 연구비는 British Heart Foundation Senior Fellowship FS/SBSRF/22/31022 (DBS), Additional Ventures Single Ventricle Fund (DBS), John Fell Fund, University of Oxford 0016065 (VSR) 및 Medical Sciences Internal Fund, University of Oxford 0016707 (VSR)로부터 제공되었습니다.

액세스가 제한되었습니다. 이 콘텐츠를 보려면 로그인하거나 체험판을 시작하세요.

재료

이 논문에 사용된 재료 목록
이름회사카탈로그 번호댓글
플라스틱 기구/유리 기구
3ml 파스퇴르 피펫, 저밀도 폴리에틸렌(LDPE), 눈금 표시됨Wheaton711116
Eppendorf Safe-lock tubes 1.5ml 마이크로튜브Eppendorf0030 120.086
Ibidi µ-18-웰 슬라이드 Ibidi81817#1.5H 유리 커버슬립, 미처리
Menzel X1000 커버슬립 22x50 mm #1.5 (0.16-0.19mm)Thermo Scientific17284904
홈이 있는 현미경 슬라이드Marienfeld Superior1320000
세이프 실 마이크로튜브 2mlSarstedt72.695.5002ml 'Eppendorf' 튜브, 라운드 바텀
용액/완충액
1x 항체 희석제/블로킹 용액Akoya BiosciencesARD1001EA
Ambion™ RnaseZap™ RNase 제염 솔루션Ambion9780.9782
시트르산 마스킹 제거 완충액Vector labs H-3300항원 마스킹 해제 용액(Antigen unmasking solution)으로 표시된,
구연산 기반의
디메틸 설폭사이드 (DMSO)Merck (Sigma-Aldrich)D8418
Hanks' Balanced Salt Solution 10xMerck (Sigma-Aldrich)H4641
과산화수소 용액Merck (Sigma-Aldrich)H1009H2O 내 30% (w/w)
메탄올Merck (Sigma-Aldrich)32213-2.5L-M
정상 당나귀 혈청 AbcamAb7475100% 스톡 농도,
최종 농도 10%
파라포름알데히드(PFA) 분말Merck (Sigma-Aldrich)158127-500g물로 4%까지 희석한다. 흄 후드 내에서 조제한다. 가열 블록 위에서 교반하며, 용해를 돕기 위해 소량의 NaOH를 첨가한다. pH 7.2-7.6
인산완충생리식염수 (PBS)Merck (Sigma-Aldrich)P4417증류수 200ml당 정제 1정
Multiplex 형광 시약 키트 v2용 RNAscope 4-Plex 보조 키트ACD (Bio-Techne)323120HRP-C4 및 HRP 블로커 함유
RNAscope™ 다중 형광 검출 키트 v2 ACD (Bio-Techne)323110Protease Plus와 Protease III를 포함함,
Protease IV, 과산화수소, TSA 완충액,
Amp1/2/3, Hrp-C1/2/3, HRP-블로커 및 DAPI
20배 SSC (Saline-Sodium-Citrate) 완충액 용액Fisher BioreagentsBP-1325-1증류수로 0.2x까지 희석하십시오.
조직 투명화 시약 Cubic-R+(M) [동물용]Tokyo Chemical IndustryT3741
트리톤™ X 100Merck (Sigma-Aldrich)X100
트윈® 20Merck (Sigma-Aldrich)P7949
초순수™ 0.5M EDTA, pH8Invitrogen15575020
1차 항체
엔도뮤신(Endomucin) 항체 (V.7C7) Santa CruzSC-654951:50 희석 비율로 사용하십시오. 
Islet 1 및 Islet 2 항체Developmental Studies 하이브리도마 뱅크34.4DS-5글리세롤로 50% 희석하십시오. 1:50 희석 비율로 사용하십시오.
PCNA (PC10) 마우스 단일클론 항체Cell Signaling Technology2586S1:500 희석 배율을 사용하십시오.
P-histone H3 (Ser10) (6G3) 마우스 단클론 항체세포 신호 전달 기술9706S1:500 희석 배율을 사용하십시오.
P-histone H3 (Ser10) (D7N8E) XP® 토끼 단클론 항체세포 신호 전달 기술53348S1:500 희석 배율을 사용하십시오.
Sox10 (E6B6I) XP® 토끼 단클론 항체 Cell Signaling Technology69661S1:500 희석 비율을 사용하십시오.
2차 항체/핵 염색제
AlexaFluor® 488-접합 Affinipure® Donkey anti-mouse IgG (H+L)Jackson Immunoresearch715-545-1511:500 희석 비율로 사용하십시오.
AlexaFluor® 647-접합 Affinipure®  Donkey anti-rat IgG (H+L)Jackson Immunoresearch712-605-1531:500 희석 배율을 사용하십시오.
AlexaFluor® 647-접합 Affinipure® F(ab')2 단편 Donkey anti-mouse IgG (H+L)Jackson Immunoresearch715-606-1501:500 희석 비율로 사용하십시오.
Cy™3 Affinipure® 돈키 항-토끼 IgG (H+L)Jackson Immunoresearch711-165-1521:500 희석 배율을 사용하십시오.
DAPIMerck (Sigma-Aldrich)D9542~에서 사용됨 1 µg/ml
RNAscope 프로브
RNAscope® 프로브 - Mm Sp5-C4ACD (Bio-Techne)405681-C4C4 프로브
RNAscope® 프로브 - Mm-Dlx2-C3ACD (Bio-Techne)555951-C3C3 프로브
RNAscope® 프로브 - Mm-Msx1ACD (Bio-Techne)421841C1 프로브
RNAscope® 프로브 - Mm-Sox10-C2ACD (Bio-Techne)435931-C2C2 프로브
RNAscope 형광체
Opal 780 시약 팩Akoya BiosciencesFP1501001KT Opal 780 시약, Opal TSA-DIG 포함
DMSO
TSA-vivid™ 형광체 520ACD (Bio-Techne)323271현재 ClariTSA™ 형광체 520
TSA-vivid™ 형광체 570ACD (Bio-Techne)323272이제 ClariTSA를 사용합니다.™ 형광체 570
TSA-vivid™ 형광색소 650ACD (Bio-Techne)323273현재 ClariTSA™ 형광체 650
장비/소프트웨어
Fluoview FV4000 레이저 스캐닝  현미경명백한FV4000들뜸 레이저: 405/488/561/640/730 nm.
검출 파장 (그림 3): 430-470, 500-540, 570-620,
650-710, 750-850 nm. 검출 파장(그림 4) 430-470,
500-531, 570-620 nm.
GraphPad Prism 10Graphpadv10.6.1
가열 블록연구비 지원금QBT1
Hybrigene 하이브리다이제이션 오븐Techne (Bibby Scientific)FHB4DD
Ixplore IX83 SpinSR 초고해상도 현미경 시스템Olympusixplore-ix83-spinsr들뜸 레이저: 405/488/561/640 nm.
방출 필터: 447/60, 525/50, 617/73, 685/40. 
Zeiss Arivis Pro Carl Zeiss Microscopy GmbHv. 4.5 (이전 Arivis Vision 4D)
Zeiss Arivis SIS 컨버터Carl Zeiss Microscopy GmbHv. 4.3.0

참고문헌

  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).

액세스가 제한되었습니다. 이 콘텐츠를 보려면 로그인하거나 체험판을 시작하세요.

재인쇄 및 허가

더 많은 논문 탐색

전체 마운트 염색단백질 발현RNAscope 염색면역형광 염색다중 검출공초점 이미징3D 분석배아 발달

이 논문이 게재되었습니다

동영상 곧 제공

문의하기
도서관에 추천하기

개인정보 처리방침

이용 약관

정책

저작권 © 2026 MyJoVE Corporation. 모든 권리 보유.