Nb100 1028

For brain tissue preparation, mice were deeply anesthetized with Zoletil (12.5 mg/kg) and Rompun mix (17.5 mg/kg) administered intraperitoneally. Mice were perfused transcardially with heparin dissolved in PBS (pH 7.2). The dissected brain tissues were frozen at -80 °C for Western blotting. Tissues were homogenized with total RNA and protein isolation kit (NucleoSpin® RNA/Protein #740933.50, Macherey-Nagel, Dűren, Germany). The protein samples were quantified with Pierce™ BCA Protein Assay Kit and 50 μg protein sample were used for each Western blot. The primary antibodies were applied in the following concentrations: anti-GFAP (rabbit, 1: 1,000; Dako #Z0334), anti-Iba1 (rabbit, 1:300; Wako #NB100-1028), anti-EAAT1 (rabbit, 1:1,000; Santacruz # sc-15316), anti-EAAT2 (rabbit, 1:1,000; Cellsignaling #3838), anti-Cx43 (mouse, 1:1,000; Santacruz #sc-59949), anti-PSD95 (mouse, 1:2,000; Thermo #MA1-046), anti-NeuN (mouse, 1:1,000; Millipore #MAB377) anti-GAPDH (rabbit, 1:10,000; Abfrontier #LF-PA0018). Secondary antibodies were conjugated with horse-radish peroxidase (HRP) (1: 10,000, Invitrogen). The HRP signals were visualized using an enhanced chemiluminescent (ECL, Abfrontier #LF-QC0101, Gyeonggi-do, Korea) substrate.

Mice were anesthetized and perfused intracardially with 0.9% saline. Knee and brain samples were collected and post-fixed for 12 h in 4% paraformaldehyde. Brain tissue sections of 20 μm thickness were acquired with a CM3050S microtome (Leica, Wetzlar, Germany). Brain tissue sections were permeabilized in 0.3% TritonX-100 containing phosphate-buffered saline (PBST), followed by blocking with 0.3% normal donkey serum and 1% bovine serum albumin for 60 min at room temperature. The sections were then incubated with ionized calcium binding adaptor molecule 1 (Iba1, microglia marker, 1:200, goat, Novus Biologicals, NB100-1028), β-amyloid (amyloid plaques marker, 1:200, mouse, Santa Cruz, sc-28365), or Neu-N (neuron marker, 1:1000, rabbit, Millipore, ABN78) as described previously [13 (link)]. Sections were mounted with DAPI (Molecular Probes, Life Technologies).