Amyloid β 1 42

Rats were deeply anesthetized by intraperitoneal injection of ketamine (87 mg/Kg) and Xylazine (13 mg/Kg) which was followed by transcardial perfusion with 0.9% saline using a Simon Varistaltic Pump. For each rat, a section of the brain (frontal cortex) tissue was snapped frozen in liquid nitrogen and stored at −80°C for western blot analysis. The rest of the brain was immersion fixed in 4% paraformaldehyde for 48 h after which the brains were embedded in paraffin and seven equally spaced coronal blocks (~2 mm) cut using a rat brain matrix, as previously described (Defazio et al., 2014 (link)). A series of adjacent 6 μm thick C sections were cut and employed for various staining. Brain coronal tissue sections were prepared and an antibody against AQP4 (Millipore, 1:1,000) was employed to assess water channel dysfunction. Antibodies against APC (oligodendrocyte marker, Genway, 1:20), NG2 (oligodendrocyte progenitor cell marker, MilliporeSigma, 1:400), Synaptophysin (synaptic protein, Abcam, 1:400) were also used. Bielschowsky silver was used to stain axons and Luxol fast blue was used to stain myelin. An antibody against amyloid-β 1–42 (amyloid-β 1–42, Abcam, 1:100) was used to assess amyloid-β levels. Similar procedures without the addition of primary antibodies were used as controls.

Protein was extracted from samples using Trizol (Invitrogen). The BCA kit (Thermo Scientific) was used to measure protein concentration and 40 μg of protein/lane loaded in a 10% SDS PAGE precast gel (Invitrogen). The gel was placed in a tank where 120 volts of electrical current was run for approximately one and a half hours. The gel was subsequently transferred to a nitrocellulose membrane using the iBlot transfer system (Invitrogen). This membrane was blocked in 2% I-Block (Applied Biosystems) in 1× TBS-T for 1 h, and then either β-actin (Abcam, 1:10,000), TGF-β (R&D Sytems, 1:1,000), Amyloid-β 1–40 (MyBioresource, 1:500) or Amyloid-β 1–42 (Abcam, 1:1,000) was used. For Synaptophysin and PSD-95, 30 μg of protein/lane was loaded, and either GAPDH (Abcam, 1:5,000), PSD-95 (Cell signaling, 1:750) or Synaptophysin (Chemicon, 1:2,000) were used. Secondary antibodies (anti-mouse, Jackson ImmunoResearch) were added at 1:1,000 dilution in 2% I-Block in 1× TBS-T at room temperature. The membranes were washed with 1× TBS-T, and then Luminol Reagent (Santa Cruz) was added. The membranes were then developed using a FluorChem E Imager system (ProteinSimple). Grayscale images were analyzed using ImageJ and normalized to β-actin or GAPDH.