小鼠胚胎主动脉-性腺-中肾（AGM）区域冰冻切片及免疫荧光染色方法

Yan Jia; Sixuan Huo; Mei Li

doi:10.21769/BioProtoc.5828

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Peer-reviewed

Protocol for Mouse Embryonic Aorta–Gonad–Mesonephros (AGM) Region Frozen Sectioning and Immunofluorescence

小鼠胚胎主动脉-性腺-中肾（AGM）区域冰冻切片及免疫荧光染色方法

YJ Yan Jia ^*

SH Sixuan Huo ^*

ML Mei Li

(*contributed equally to this work) 发布: 2026年09月20日第16卷第18期 DOI: 10.21769/BioProtoc.5828 浏览次数: 59

评审: Jessica DavisRitika GhosalSrinivasan MahalingamAnonymous reviewer(s)

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Abstract

The aorta–gonad–mesonephros (AGM) region is the site where hematopoietic stem cells (HSCs) first emerge during development, and is therefore widely used to study in vivo hematopoiesis and to discover novel regulatory mechanisms. The endothelial-to-hematopoietic transition (EHT) process can be directly observed via immunofluorescence on frozen sections of the AGM region. However, the mouse AGM region is extremely delicate and lies deep within the embryo, between the notochord and the somatic mesoderm. Here, we present a step-by-step protocol covering embryo collection, fixation, dehydration, and embedding with a defined orientation, followed by frozen sectioning, immunofluorescence staining, and confocal imaging. The protocol is highly reproducible and easy to follow and provides clear instructions on orienting the embryo and anatomically locating AGM. By filling a technical gap, the protocol can enable researchers to reliably study HSC emergence and EHT in the mouse embryonic AGM.

Key features

• Provides detailed instructions on embryo embedding orientation and microscopic observation during cryosectioning to precisely locate the mouse AGM region.

• Covers all steps from embryo harvesting, cryosectioning, and immunofluorescence to confocal imaging, ensuring high reproducibility.

• Developed for delicate mouse embryonic AGM tissue (e.g., E9.5–E11.5), enabling direct observation of EHT via immunofluorescence.

Keywords: Aorta–gonad–mesonephros (主动脉-性腺-中肾)

Hematopoietic stem cells (造血干细胞)

Endothelial-to-hematopoietic transition (内皮向造血转化)

Cryosectioning (冰冻切片)

Immunofluorescence (免疫荧光)

Graphical overview

Graphical overview of immunofluorescence staining and imaging of E10.5 mouse aorta–gonad–mesonephros (AGM) cryosections

Background

Hematopoietic stem cells (HSCs) reside at the apex of the hematopoietic hierarchy, possessing the capacity for self-renewal and differentiation into all downstream blood cell lineages [1–3]. HSCs are generated during definitive hematopoiesis in the embryonic stage in mice, arising from a specialized population of hemogenic endothelial cells (HECs) in the aorta–gonad–mesonephros (AGM) region through endothelial-to-hematopoietic transition (EHT) [4,5]. During this process, HECs give rise to immature hematopoietic cells, including pre-HSCs and hematopoietic progenitors. These cells subsequently form intra-aortic hematopoietic clusters (IAHCs) attached to the aorta [4,6]. Elucidating the cellular and molecular mechanisms underlying HSC origination in the AGM region is of great significance for understanding normal hematopoietic development and for guiding the induction of HSCs from pluripotent stem cells in vitro [7,8].

Previous studies on the AGM region have systematically revealed the dynamic stages of hematopoietic development and key molecular markers, in which immunofluorescence has played an indispensable role. For example, this technique enables the identification of intermediate states of hematopoietic development, such as the discovery and in situ localization of VE-cadherin⁺CD45⁺ type II pre-HSCs [9]. By co-staining with the hematopoietic transcription factor RUNX1 and the endothelial markers CD31 or VE-Cadherin, immunofluorescence further allows the characterization of various hematopoietic molecules in the aorta. For instance, angiotensin-converting enzyme (ACE) was identified as a marker of HECs, while CD44 serves as a marker of EHT [10,11]. Also, immunofluorescence can be used to validate the specific expression pattern of newly generated fluorescent reporter mice (e.g., Gfi1:H2B-Tomato and Runx1+23GFP) in the AGM region, and to morphologically determine the intermediate state of the transgenic-labeled hematopoietic precursors [12,13]. Moreover, this technique has been applied to assess the effects of genetic perturbations on hematopoietic development. For example, after endothelial-specific deletion of Meis1, Meis1^flox/flox;VEC-Cre;Runx1+23GFP embryos at embryonic day (E) 10.5 were analyzed by GFP and CD31 expression in the dorsal aorta, and the number of IAHCs was quantified to evaluate hematopoietic defects [14]. In summary, frozen-section immunofluorescence provides clear and single-cell resolution visualization for studying HSC emergence in the AGM region. It complements flow cytometry, in vitro functional assays, and single-cell transcriptomic sequencing, and thus possesses irreplaceable value in this field.

Although frozen-section immunofluorescence of the AGM region has been used in numerous studies, and its basic procedures have been described, critical details for precisely locating the AGM region—such as the optimal embryo orientation for embedding and how to identify AGM-containing sections during cryosectioning—have not been thoroughly addressed. The lack of such details makes it difficult for beginners to consistently obtain the required sections, leading to relatively poor experimental reproducibility. This protocol provides, for the first time, systematic guidance on embryo collection, embedding, anatomical landmark recognition during cryosectioning, and confocal imaging. Therefore, it can effectively improve section consistency and reproducibility, reduce waste of embryos and time, and offer a reliable methodological foundation to advance AGM-related hematopoietic research.

Materials and reagents

Biological materials

1. E9.5, E10.5, and E11.5 wild-type C57BL/6 mouse embryos obtained from timed matings (bred in-house)

2. E10.5 C57BL/6 Cdh5-Cre; Rosa26-CAG-loxP-STOP-loxP-tdTomato reporter mouse embryos obtained from timed matings of mice bred in-house (Cdh5-Cre mice were purchased from Cyagen Biosciences; Rosa26-CAG-loxP-STOP-loxP-tdTomato mice were generated by Cyagen Biosciences; used in Figure S2)

Reagents

1. α-minimum essential medium (α-MEM) (Cytiva, catalog number: SH30265.01)

2. Fetal bovine serum (FBS) (Thermo Fisher Scientific, Gibco, catalog number: A5256701)

3. Phosphate buffered saline (PBS), 10× (M&C Gene Technology, catalog number: CC008.1)

4. 4% paraformaldehyde (PFA) (Servicebio, catalog number: G1101)

5. Sucrose (Sinopharm, catalog number: 10021418)

6. Triton X-100 (Diamond, catalog number: A110694)

7. 75% ethanol (Sinopharm Chemical Reagent, catalog number: 80176965)

8. DAPI (Beyotime, catalog number: C1002)

9. Anti-fade mounting medium (Invitrogen, catalog number: P36984)

10. Tissue-Tek O.C.T. compound (Sakura Finetek, catalog number: 4583)

11. Rat anti-CD31/PECAM-1 primary antibody (Santa Cruz, catalog number: sc-18916)

12. Mouse anti-RUNX1 primary antibody (Santa Cruz, catalog number: sc-365644)

13. Alexa Fluor 488-conjugated goat anti-mouse secondary antibody (Invitrogen, catalog number: A28175)

14. Alexa Fluor 546-conjugated goat anti-rat secondary antibody (Invitrogen, catalog number: A11081)

Solutions

1. 10% FBS/α-MEM (see Recipes)

2. 10% FBS/PBS (see Recipes)

3. 15% sucrose/PBS (see Recipes)

4. Immunofluorescence blocking buffer (blocking buffer) (see Recipes)

5. Antibody dilution buffer (see Recipes)

Recipes

1. 10% FBS/α-MEM

Reagent	Final concentration	Volume
α-MEM	N/A	45 mL
FBS	10%	5 mL
Total	N/A	50 mL

Thaw FBS at 4 °C overnight. Prepare 45 mL of α-MEM in a sterile 50 mL conical tube. Add 5 mL of FBS to the α-MEM and mix gently by inversion. Avoid vigorous shaking to prevent foaming. The 10% FBS/α-MEM solution can be stored at 4 °C for up to one week.

Note: Heat inactivation of FBS is not necessary.

Caution: Freeze FBS in aliquots to avoid freeze/thaw cycles and store the aliquots at -20 °C.

2. 10% FBS/PBS

Reagent	Final concentration	Volume
FBS	10%	5 mL
10× PBS	1×	5 mL
Autoclaved distilled water	N/A	40 mL
Total	N/A	50 mL

Thaw FBS at 4 °C overnight. Prepare 40 mL of autoclaved distilled water in a sterile 50 mL conical tube. Add 5 mL of 10× PBS and mix thoroughly. Add 5 mL of FBS to the PBS and mix gently by inversion until homogeneous. The 10% FBS/PBS solution can be stored at 4 °C for up to one week.

3. 15% sucrose/PBS

Reagent	Final concentration	Quantity
Sucrose	15% w/v	7.5 g
10× PBS	1×	5 mL
Autoclaved distilled water	N/A	Up to 50 mL
Total	N/A	50 mL

Prepare approximately 35 mL of autoclaved distilled water in a sterile 50 mL conical tube. Add 5 mL of 10× PBS and mix thoroughly. Weigh 7.5 g of sucrose and add it to the tube. Mix gently at room temperature until the sucrose is completely dissolved. Adjust the final volume to 50 mL with autoclaved distilled water and mix well. The 15% sucrose/PBS can be stored at 4 °C for up to one week.

4. Blocking buffer

Reagent	Final concentration	Volume
10× PBS	1×	10 mL
FBS	10%	10 mL
Triton X-100	0.2%	0.2 mL
Autoclaved distilled water	N/A	79.8 mL
Total	N/A	100 mL

Prepare 79.8 mL of autoclaved distilled water in a sterile container. Add 10 mL of 10× PBS and mix thoroughly. Add 10 mL of FBS and mix gently until homogeneous. Add 0.2 mL of Triton X-100 and mix slowly to avoid foaming. The blocking buffer is recommended to be prepared fresh and used immediately.

5. Antibody dilution buffer

Reagent	Final concentration	Volume
10× PBS	1×	10 mL
FBS	3%	3 mL
Triton X-100	0.2%	0.2 mL
Autoclaved distilled water	N/A	86.8 mL
Total	N/A	100 mL

Prepare 86.8 mL of autoclaved distilled water in a sterile container. Add 10 mL of 10× PBS and mix thoroughly. Add 3 mL of FBS and mix gently until homogeneous. Add 0.2 mL of Triton X-100 and mix slowly to avoid foaming. The antibody dilution buffer should be prepared fresh and used immediately.

Laboratory supplies

1. Sterile 50 mL conical tubes (CellPro, catalog number: 801501)

2. Sterile 15 mL conical tubes (CellPro, catalog number: 801151)

3. Sterile 35 mm cell culture dishes (Thermo Fisher Scientific, catalog number: 150460)

4. 60 mm glass culture dish (Changde BKMAM, catalog number: 120403007)

5. Adhesion microscope slides (Citotest, catalog number: 188105)

6. Coverslips (Citotest, catalog number: 10212450C)

7. Humidified chamber (Sangon Biotech, catalog number: E678020)

8. Light-protective staining box (Nantong Supin, for 24 slides)

9. Hydrophobic barrier pen (Daido Sangyo, catalog number: 0010G)

10. Lint-free wipes (Kimtech, catalog number: 34155)

11. Aluminum foil (Labshark, catalog number: 130410222)

12. Aluminum foil molds (in-house)

13. 31-gauge (G) insulin syringe (Shanghai Kindly, catalog number: U-40)

14. Iris forceps (Sangon Biotech, catalog number: F519023)

15. Iris scissors (Sangon Biotech, catalog number: F519232)

16. Size 5 fine forceps (Fine Science Tools, catalog number: 11254-20)

17. Cryostat specimen chucks (Leica Biosystems, catalog number: 14037008587)

18. Cryostat blades (Leica Biosystems, catalog number: 14035838925)

19. Micropipettes, 0.5–10, 20–200, and 100–1,000 μL (Eppendorf, catalog numbers: 3123000020, 3123000055, and 3123000063)

20. Pipette tips, 0.1–10, 2–200, and 50–1,000 μL (Biosharp, catalog numbers: BS-10-T, BS-200-T, and BS-1000-T)

21. Sterile disposable serological pipets, 5, 10, and 25 mL (Corning, catalog numbers: CLS4487, CLS4488, and CLS4489)

22. Pipette controller, 0.1–100 mL (Eppendorf, catalog number: 4430000018)

23. Glass graduated cylinders, 50 and 100 mL (SolelyBio, catalog numbers: SBM0085 and SBM0233)

24. Glass beakers, 100 and 250 mL (SolelyBio, catalog numbers: SBM0084 and SBM0382)

25. Glass reagent bottles with screw caps, 250, 500, and 1,000 mL (Shuniu, catalog numbers: SN03250, SN03500, and SN031000)

Equipment

1. Stereomicroscope (Nikon, model: SMZ745T)

2. Cryostat (Leica Biosystems, model: CM1860)

3. Confocal laser scanning microscopes (Zeiss, model: LSM900)

4. -80 °C freezer (Haier, model: DW-86L486)

5. 4 °C refrigerator (Haier, model: BCD-520WDPD)

6. 37 °C incubator (Shanghai Zhicheng, model: ZXGP-B2080)

7. Orbital shaker (Changzhou Nuoji, model: KTS-103H)

8. Liquid nitrogen storage container (Chengdu Jinfeng, model: YDS-175-216)

9. Carbon dioxide (CO₂) euthanasia chamber (Braintree Scientific, model: COIC)

Software and datasets

1. ZEN (blue edition) microscopy software (Zeiss, version: 3.6)

2. Imaris x64 (Oxford Instruments/Bitplane, version: 9.0.1)

Procedure

English

中文翻译

文章信息

稿件历史记录

提交日期: Jun 28, 2026

接收日期: Aug 20, 2026

在线发布日期: Sep 11, 2026

出版日期: Sep 20, 2026

版权信息

如何引用

Jia, Y., Huo, S. and Li, M. (2026). Protocol for Mouse Embryonic Aorta–Gonad–Mesonephros (AGM) Region Frozen Sectioning and Immunofluorescence. Bio-protocol 16(18): e5828. DOI: 10.21769/BioProtoc.5828.