Biotinylated donkey anti rabbit

Sections were placed in 0.01M phosphate buffered saline (PBS) solution with 2% sodium azide added and 0.3% hydrogen peroxide for 15 min. After three 5-min washes with PBS, sections were transferred to PBS with 0.3% Triton added (PBST) containing 2% normal donkey serum (NDS) for 2 h. Sections were incubated overnight (16h) in the same solution with the addition of primary antibody (Fos antibody at 1:10,000 Calbichem, Santa Cruz, CA). Sections were rinsed three times (3x) in PBST and transferred to secondary antibody (biotinylated donkey anti-rabbit, 1:500, Jackson ImmunoResearch Laboratories, West Grove, PA) for 2 h. Next, sections were rinsed 3x in PBST and transferred to solution containing avidin-biotin complex (1:500, Vector Laboratories, Burlington, CA) for 1.5 h. Sections were rinsed twice with PBST and once with 0.05 M Tris buffer for 5 min. To visualize Fos-related antigen-positive cells, sections were placed in 3′, 3′-diaminobenzidine (DAB) with 0.0002% H₂O₂ and 0.6% nickel ammonium sulphate in 0.05 M Tris buffer for 4.5 min. The DAB reaction was stopped by immediately transferring sections into Tris buffer. Next, sections were rinsed 3x in PBS for 5 min and transferred to PBS-Az for 45 min.

The antibodies used in the present study are provided in Table 1. Secondary antibodies including biotinylated donkey anti-rabbit (Jackson Immunoresearch, West Grove, PA, USA) (catalog number: 711-065-152), and Alexa fluor 488-conjugated goat anti-rabbit (Life Technologies, Grand Island, NY, USA) (Ref# A11034), horseradish peroxidase-conjugated goat anti-rabbit and anti-mouse (Santa Cruz Biotechnology, TX, USA), (sc-2005 and sc-2004)) were used. The test drug; BSB (catalog number: 14462), ROT (catalog number: R8875), glutathione (GSH) assay kit (catalog number: CS0260) and other analytical grade reagents used in this study were obtained from Sigma Aldrich, St. Louis, MO, USA.

IHC. Tissue was processed for GFP, mCherry, dsRed and/or c-Fos (FOS-IR) IHC as previously described (D’Agostino et al., 2016 ). Briefly, mice were transcardially perfused with phosphate buffered saline (PBS) followed by 10% neutral buffered formalin (Sigma-Aldrich). Brains were extracted, post-fixed in 10% neutral buffered formalin at 4°C, cryoprotected in 20% sucrose at 4°C and then sectioned coronally on a freezing sliding microtome at 25 μm. Tissue was processed for chicken anti-GFP (1:1000; AbCam, ab13970), rabbit anti-dsRED (1:1000; Rockland, 600-401-379), goat anti-mCherry/RFP (1:1000; Sicgen, AB0040-200) or anti-c-FOS (1:5000, rabbit, Calbiochem, PC38) primary antibodies and a biotinylated donkey anti-rabbit (1:500, Jackson ImmunoResearch Laboratories, Inc.) or Alexa Fluor (1:500, Life Technologies) secondary antibodies using standard protocols previously described (Lam et al., 2009 (link), Heisler et al., 2006 (link)). Tissue was then mounted on slides, cover slipped and the NTS visualized using an Axioskop II microscope (Carl Zeiss, Oberkochen, Germany) and Adobe Photoshop CS5 software. Images of single-label immuoreactivity (IR) for GFP or mCherry were used to visualize and analyze NTS injection sites.