Biotinylated anti rabbit secondary antibody

The primary antibodies used were Collagen IV (1:400 dilution; Cosmo Bio, Catalog Number LSL-LB1403) and CD13 (1:100 dilution; R&D Systems, Catalog Number AF2335), NeuN (1:200 dilution; Millipore Sigma, Catalog Number MAB377 Clone A60), Iba 1 (1:200 dilution; FUJIFILM Wako Chemicals, Catalog Number 019-19741). Mice were sacrificed by decapitation, and brains were dissected. Brains were postfixed by immersion in Accustain (Sigma Aldrich). After fixation, brains were cryoprotected in 30% sucrose and cut into 60 μm thick sections with a cryostat. Free-floating coronal brain slices were subjected to a protein block at room temperature for 30 min. Slices were then incubated with primary antibodies at their respective concentrations overnight at 4°C, followed by consecutive incubations of biotinylated anti-rabbit secondary antibody (1:200; Jackson Immunoresearch) and biotinylated anti-goat secondary antibody (1:200; Jackson Immunoresearch) both for 1 h at room temperature. DyLight fluorophores (Jackson Immunoresearch) were added after each secondary incubation. Slices were then mounted onto glass slides under coverslips using Fluoro-Gel II mounting media with DAPI (Electron Microscopy Services).

The antibodies used in this study included polyclonal rabbit anti-tyrosine hydroxylase (Novus Biologicals, Littleton, CO, USA), polyclonal rabbit anti-inducible nitric oxide synthase (iNOS), anti-cyclooxygenase-2 (COX-2), and anti-glial fibrillary acidic protein (GFAP) (Abcam, Cambridge, MA, USA), polyclonal rabbit anti-ionized calcium binding adaptor molecule-1 (Iba-1) (Wako Chemicals, Richmond, VA, USA), biotinylated secondary anti-rabbit antibody (Jackson Immunoresearch, West Grove, PA, USA), and Alexa fluor 488-conjugated goat anti-rabbit secondary antibodies (Life Technologies, Grand Island, NY, USA). The test compound, thymol was procured from Santa Cruz Biotechnology Inc, CA, USA. ROT, the chemical to induce PD in rats were purchased from Sigma Aldrich, St. Louis, MO, USA. The ELISA assay kits for antioxidant enzymes and glutathione (GSH) as well as other analytical grade reagents were also obtained from Sigma Aldrich, St. Louis, MO, USA.

Polyclonal rabbit anti-cyclooxygenase-2 (COX-2), anti-inducible nitric oxide synthase (iNOS), and anti-glial fibrillary acidic protein (GFAP) antibodies were purchased from Abcam, Cambridge, MA, USA. Anti-ionized calcium-binding adaptor molecule-1 (Iba-1) polyclonal rabbit antibody was procured from Wako Chemicals, Richmond, VA, USA. Polyclonal rabbit anti-tyrosine hydroxylase antibody was obtained from Novus Biologicals, Littleton, CO, USA. Alexa fluor 488 conjugated secondary goat anti-rabbit antibodies were purchased from Life Technologies, Grand Island, NY, USA. Biotinylated secondary anti-rabbit antibody was purchased from Jackson Immunoresearch, West Grove, PA, USA. Nerolidol was purchased from Santa Cruz Biotechnology Inc., CA, USA. ROT and the assay kit for reduced glutathione (GSH) and other reagents of analytical grade were purchased from Sigma-Aldrich, St. Louis, MO, USA.

MO3.13 cells were plated on glass cover slips (25000 cells/cm²) into 24-well plates for 2 days and then treated with serum-free medium. After 24 h of treatment, cells were fixed for 30 min in 2% paraformaldehyde, permeabilised with 0.1% Tween-20 and blocked with 5% horse serum for 15 min. Cells were then incubated overnight with anti-myelin basic protein antibody (1 : 50, AbCam ab62631). Cover slips were washed with PBS (10 min for three times), incubated with anti-rabbit biotinylated secondary antibody (Jackson Immuno Research Europe, Suffolk, UK, 1 : 500) for 90 min, washed with PBS and then stained with Alexa 488 Streptavidin (Molecular Probes, Netherlands, 1 : 1000) for 30 min. Finally, the cover slips were mounted onto glass microscope slides in presence of Vectashield mounting medium with DAPI (Vector Laboratories inc. Burlingame, CA, USA). Confocal images were acquired as z-stacks by using a scanned on z axis with a SP2 TCS-NT Leica (Nussloch, Germany) laser scanning confocal microscope, equipped with 40 × 1.2 NA Plan Apo oil objective. The optical slice was set to 1 μM (FWHM).

At 24 weeks of age, animals were processed for histological assessment. Following a terminal dose of pentobarbitone (100 mg/kg; Virbac, Peakhurst, Australia) animals were transcardially perfused with 50 mL 0.2 M phosphate buffered saline and 250 mL paraformaldehyde (PFA, 4% in 0.2 M phosphate buffer with 0.2% picric acid). The brains were collected and post-fixed in PFA for 2 h, followed by cryo-protection in 20% sucrose PBS solution for one to two days. The brains were snap frozen on dry ice and coronal sections were collected in series at 40 µm using a freezing-microtome (Leica, Wetzlar, Germany).
Immunohistochemistry was performed on free-floating sections as previously described [54 (link)]. Primary antibodies specific for NeuN (1:500; Abcam, ab104225, Cambridge, MA, USA) or Iba1 (1:1000; Wako, West Grove, PA, USA, 019-19741) were incubated overnight to detect neurons or microglial, respectively. After washing, the tissue was incubated with anti-rabbit biotinylated secondary antibody (1:400, Jackson ImmunoResearch, 711-065-152, USA) with 2% donkey serum for 2 h, followed by washing and incubation in avidin-biotin complex (ABC Elite kit, Vectastain; Vector Laboratories, Burlingame, CA, USA) for 1 h. A peroxidase-driven precipitation of diaminobenzidine (DAB) was used to visualise chromogenic labelling, catalysed with a 1% H₂O₂ solution and terminated by washing in PBS.