Biotinylated anti goat secondary antibody

For the immunohistochemistry, mother and pup-deprived rats (3-3) were deeply anesthetized and fixed with 4% paraformaldehyde via transcardially perfusion. Brains were removed and postfixed in 4% paraformaldehyde for 24 h then transferred to 20% sucrose in phosphate buffer (PB, pH=7.4) for 48 h. Serial coronal brain sections were cut at 40 µm on a sliding microtome. Sections were collected in PB containing 0.05% sodium-azide and stored at 4 o C. The mPFC sections were incubated in anti-Cryab primary antibody (1:20 dilution) followed by a biotinylated anti-goat secondary antibody (1:1,000 dilution, Jackson Immunoresearch, West Grove, PA). Visualization was performed using an ABC kit (1:500 dilution, Vector Laboratories, Burlingame, CA, USA) and a Ni-DAB reaction. For double labeling, the visualization of Cryab was performed using FITC-tyramide (1:8,000)
A C C E P T E D M A N U S C R I P T amplification followed by incubation with mouse anti-calbindin (1:900 dilution, catalogue number: C9848, Sigma) or anti-parvalbumin (1:800 dilution, catalogue number: P3088, Sigma) antibodies, which were visualized with Alexa 594-conjugated anti-mouse IgG (1:400 dilution, Thermo Fisher Scientific, Waltham, MA). Subsequently, the sections were mounted, dried, and coverslipped in antifade medium (Prolong Antifade Kit; Molecular Probes).