Procedures

  Immunohistochemistry technique

  Preparation of fresh frozen sections

E16.5 whole embryos from pregnant ICR mice were embedded and frozen in OCT compound (SAKURA Finetechnical, Tokyo, Japan), and stored at -80ºC until use. Frozen samples were sectioned at 7-10 μm thickness and placed on MSA-coated glass slides (MATSUNAMI, Japan).
Experiments and animal care have been performed in compliance with Aichi Medical University and Osaka University institutional animal care.

  Standard immunostaining procedures

1. Frozen sections were air-dried and treated with fixing reagent^*1 at room temperature, and then washed three times with PBS.
2. Options: In some cases, sections were treated with a mixture of hyaluronidase and chondroitinase ABC for 30 min at room temperature and/or 0.1M KCl-HCl (pH 1.5) for 0.5-10 min at room temperature before blocking with the serum^*2
3. Treated sections with 0.3% H₂O₂/0.3% goat serum^*3/PBS for 15 min and then washed three times with PBS.
4. Blocked 60 min in 2% goat serum^*3/PBS.
5. Incubated with primary antibody diluted in 0.1% goat serum^*3/PBS.
6. Washed four times in PBS.
7. Incubated with secondary antibody conjugated with horseradish peroxidase for 60 min at room temperature.
8. Washed four times in PBS.
9. Added DAB solution to sections and incubated for appropriate time at room temperature.
10. Washed with PBS four times.
11. Counterstained with hematoxylin by the procedure described elsewhere.
12. Dehydrated with graded alcohols and xylene and then applied coverslips with mounting medium.

^{*1, 2} Conditions for each staining are indicated in the "Antibody information" page.
^*3 Calf or fetal bovine serum was used for a goat primary antibody. In some cases, mouse serum was used.