Anti fading media

Immunohistochemistry was performed on hAPP-J20 AD mouse brains and compared to WT littermate controls. Brains were fixed in freshly prepared 4% PFA and cut into 40 μm-thick vibratome sections. Immunolabeling was performed using mouse monoclonal antibodies against neuronal-specific proteins: microtubule associated protein 2 (MAP2, 1:100; Millipore) and synaptophysin (1:500; Millipore). After overnight incubation with primary antibody, sections were incubated with Texas red or FITC-conjugated horse anti-mouse IgG secondary antibody (1:75; Vector Laboratories) and mounted with anti-fading media (Vector). Immunosignals were analyzed by quantitative immunofluorescence using blind-coded sections, serially imaged on a laser-scanning confocal microscope, and quantified using NIH Image 1.43 software. At least three sections for each brain and four fields for each section were analyzed in each brain area.

Neuropathological analysis was performed using methods previously published by our group (Talantova et al. 2013 (link); Meng et al. 2011 (link); Cho et al. 2011 (link)). To determine the effects of cyanide exposure and CA treatment, brains were fixed in 4% PFA, cut into 40 μm-thick vibratome sections and immunolabeled with mouse monoclonal antibodies against the neuronal-specific proteins, MAP2 (1:100; Millipore), NeuN (1:500; Millipore), and synaptophysin (1:500; Millipore). Primary antibodies were detected with horse anti-mouse FITC-conjugated antibody (1:75; Vector) and mounted under glass coverslips using anti-fading media (Vector). All sections were processed by standardized conditions. Staining was analyzed by quantitative confocal immunofluorescence using blind-coded sections, serially imaged on a laser-scanning confocal microscope, and quantified using NIH Image 1.43 software. A total of three sections for each brain and four fields for each section were analyzed in each brain area. For MAP2 and synaptophysin staining, results are expressed as percent area (% area) of the neuropil occupied by immunoreactive dendrites or terminals. NeuN-positive nuclei were counted stereologically using Stereo Investigator software (MicroBrightField, Wiliston, VT).