Avidin biotin peroxidase complex

Formalin‐fixed paraffin‐embedded tissues were sectioned and mounted on slides. After deparaffinization in xylene, rehydration, and antigen retrieval in citrate buffer solution (10 μm), sections were blocked with 3% hydrogen peroxide for 25 min. Then the sections were blocked by 10% BSA and incubated with primary antibodies overnight at 4°C. The sections were subsequently incubated with the avidin‐biotin‐peroxidase complex (Thermo Fisher Scientific, Inc.) for 25 min. The nuclei were counterstained with Mayer's hematoxylin solution (Sigma‐Aldrich, St. Louis, MO, USA).

For single labeling, brain sections were incubated with anti‐rabbit PAX6 (1:200) overnight at 4°C, and then incubated with the corresponding biotinylated secondary antibody and avidin‐biotin‐peroxidase complex (Vector Laboratories Inc., Burlingame, CA, USA). Immunoreactivity was detected with 0.05% diaminobenzidine (DAB; Sigma‐Aldrich, St. Louis, MO, USA) in Tris‐HCl buffer (0.1 M, pH 7.6) containing 0.03% H₂O₂.
For double labeling, brain sections were incubated with anti‐rabbit PAX6 overnight at 4°C, and then with rabbit secondary antibody and avidin‐biotin‐peroxidase complex (Vector Laboratories Inc., Burlingame, CA, USA). Immunoreactivity was detected with the Vectastain ABC‐AP kit (Vector Laboratories Inc., Burlingame, CA). Then, brain sections were washed and incubated with anti‐mouse GFAP (1:200; catalog MS‐1376‐P; Thermo Fisher Scientific, Waltham, MA, USA) overnight at 4°C, and subsequently with the mouse secondary antibody and avidin‐biotin‐peroxidase complex, and detected using DAB. Incubation without primary antibody served as the negative control, and no positive signal was detected.