Biotinylated secondary anti rabbit igg

Immunohistochemistry was performed as previously described (22 (link)). Mice were transcardially perfused with saline followed by 4% paraformaldehyde in phosphate-buffered saline. The perfused brains were dissected, post-fixed overnight, and then cut into 40 µm coronal sections on a vibratome (Leica). Free-floating sections were blocked with serum for 1 h and incubated overnight at 4°C with the primary rabbit polyclonal antibody for Iba-1 (ionised calcium-binding adaptor molecule 1, Cat# 019-19741; Wako). Immunohistochemistry was then performed using biotinylated secondary anti-rabbit IgG (Vector Laboratory), avidin-biotinylated peroxidase complex (Vector Laboratory), and 3,3′-diaminobenzidine (Sigma-Aldrich; Merck KGaA). The occupied areas of Iba-1 positive cells were measured in the cerebral cortex, hippocampus, and dorsal striatum. Images were taken at one or two optical planes per section at ×200 magnification (Olympus Corporation; 10× ocular and 20× objective). Qualitative evaluations of immunoreactivity were performed in a blinded manner.

Immunohistochemistry was conducted as described previously [35 (link)]. Briefly, 30 min after the last L-DOPA injection, the brains were removed, post-fixed overnight, and then cut into 40-µm coronal sections with a vibratome (Vibratome VT1000A, Leica Microsystems GmbH, Wetzlar, Germany). Free-floating sections were incubated in phosphate buffer saline (PBS) containing 3% hydrogen peroxide (H₂O₂) (v/v), rinsed 3 times in PBS, and blocked with 5% horse serum (HS) for 1 h at room temperature. Sections were incubated overnight at 4℃ with the primary antibodies. The primary antibodies were rabbit polyclonal antibodies for TH (Pel-Freez, RRID:AB_461064). After washing, the sections were incubated with biotinylated secondary anti-rabbit IgG (Vector Laboratories, Inc., Burlingame, CA, USA), which was followed by the avidin-biotinylated peroxidase complex (ABC kit, Vector Laboratories, Inc., PK6100) and 3,3'-diaminobenzidine (DAB; Sigma-Aldrich Co. LLC, D5637). Sections containing the SNc at AP -3.0 to -3.6 mm from the bregma were selected, and immunoreactive cells from the lesioned and unlesioned SNc were counted under a microscope. Qualitative evaluations of the immunoreactive cells were performed in a blind manner in terms of genotype and treatment by following the procedure introduced by Park et al. (2014) [33 (link)].