Immunohistochemistry protocol for analyzing Hedgehog pathway factors in mouse embryos

1. Determination of pregnancy in mouse was made by the observation of a vaginal plug on day 0.5 of gestation.
2. Mouse embryos at E10.5 were fixed using MEMFA (2 mM EGTA, 1 mM MgSO₄, and 3.7% formaldehyde in 100 mM MOPS; pH 7.5) overnight at 4°C.
3. The fixed embryos were immersed in 30% trehalose in 20 mM HEPES (pH 7.5) to cryoprotect the tissues overnight at 4°C.
4. They were embedded in Tissue-Tek O.C.T compound (Sakura Finetek Japan, Tokyo, Japan) and frozen immediately.
5. Frozen sections (7 µm thick) from the transverse plane were prepared using a cryostat.
6. The sections were antigen-retrieved by an ImmunoSaver (1:200 dilution, Nisshin EM, Tokyo, Japan) for 60 min at 80°C.

7. The sections were washed with 20 mM HEPES-100 mM NaCl (pH 7.5; HEPES buffer) and incubated with 10% (v/v) fetal bovine serum and 0.4% (v/v) Triton X-100 in HEPES buffer (blocking buffer) for 60 min at room temperature.

8. Reaction with primary antibodies was performed at an appropriate dilution (Table 1) with blocking buffer overnight at 4°C.
9. The sections were washed with HEPES buffer three times.
10. Reaction with secondary antibodies was performed at an appropriate dilution (Table 1) with blocking buffer for 2 h at 4°C.
11. The sections were washed with HEPES buffer three times.

12. The sections were enclosed in VECTASHIELD Mounting Medium (Vector Laboratories, Burlingame, CA, USA) containing 4,6′-diamidino-2-phenylindole dihydrochloride (DAPI) and observed under a BZ-X800 fluorescence microscope (KEYENCE, Osaka, Japan).

     

    Table 1

    

     

    

    Immunohistochemical images of the localization patterns of FOXA2, NKX2.2, OLIG2, NKX6.1, and PAX6. Nuclei were stained with DAPI (blue). Scale bars, 50 μm.
    
    
    
     

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