Vectastain abc ihc kit

Brain tissues were fixed by perfusion with 4% paraformaldehyde (PFA), transferred to a 30% sucrose solution, and prepared in to 40 μm sections using a freezing microtome. The sections and neurons were blocked with BlockAid™ blocking solution (Invitrogen, Carlsbad, CA, USA) for 1 h and then immunostained using GLP-1R (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), Iba-1 (Abcam), GFAP (Cell signaling Technology, Danvers, MA, USA), MBP (Invitrogen), TBR1 (Abcam), CTIP2 (Abcam), SATB2 (Abcam), MAP2 (Cell signaling Technology, Millipore), and 4G8 (BioLegend, San Diego, CA, USA) antibodies at 4 ℃ overnight. The sections were then incubated with Alexa Flour 488 (Invitrogen) or Alexa Flour 594 (Invitrogen) conjugated secondary antibodies for 1 h at room temperature (RT). DAPI (Vector laboratories, Burlingame, CA, USA) was used for nucleus staining. The analysis was performed using a Zeiss LSM710 confocal microscope (Carl Zeiss, Göttingen, Germany). For immunohistochemistry, we used a Vectastain ABC IHC kit (Vector Laboratories, Burlingame, CA, USA) and Nikon Eclipse E600 microscope with a DS-Fi2 digital camera (Nikon, Melville, NY, USA).

To evaluate hepatic 4-HNE adducts, a cross-section (5 µm) of the left lateral lobe of the liver was sliced and stained by rabbit polyclonal anti-4-HNE (Abcam, Cambridge, MA, USA) and goat anti-rabbit polyclonal antibodies (Vectastain ABC IHC kit, Vector Laboratories Inc., Burlingame, CA, USA).

A cross section of the liver was sliced at 5 µm and stained using rabbit polyclonal anti-4-HNE (Abcam, Cambridge, MA, USA) and goat anti-rabbit polyclonal secondary antibodies (Vectastain ABC IHC kit; Vector Laboratories, Burlingame, CA, USA) to evaluate 4-HNE adducts. Overall, liver lipid peroxidation was determined by a thiobarbituric acid reactive substrate (TBARS) assay as described by Volpi and Tarugi [44 (link)]. The liver lysate was mixed with 0.2% thiobarbituric acid in 2 M sodium acetate buffer containing 5% butylated hydroxytoluene. The mixtures were incubated at 95 °C for 45 min followed by centrifugation. The supernatant was injected into high performance liquid chromatography (HPLC), equipped with a fluorescence detector (FLD-3100; Thermo Scientific, Sunnyvale, CA, USA) and a 5 µm Symmetry C18 reversed phase column (4.6 mm × 150 mm; Eka Chemicals, Bohus, Sweden). The complex of MDA and thiobarbituric acid was monitored by fluorescence detection with excitation at 515 nm and emission at 553 nm.