Pa5 19468

Proteins have been isolated from a complete cross section of the OCT-embedded cerebrum at the level of the hippocampus. Cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (1 mM phenylmethylsulfonyl fluoride, 1% sodium deoxycholate, 50 mM Tris–HCl [pH 7.4], 1% Triton X-100, 0.1% SDS, 150 mM NaCl) with added protease inhibitor cocktail (cOmplete, EDTA free; Roche Diagnostics GmbH, Mannheim, Germany). The total protein concentration of each sample was determined using the Pierce™ BCA™ Protein-Assay (Thermo Fisher, Rockford, IL, USA) according to the manufacture’s instructions. 20 µg of total protein were separated by SDS–polyacrylamide gel electrophoresis followed by transfer to polyvinylidene difluoride (PVDF) membranes (Novex™). Rabbit polyclonal antibodies were used to detect IL-1β (P420B; Thermo Fisher; 1:1000), IL-18 (PA5-79481; Thermo Fisher; 1:1000), and Laminin (PA5-19468; Thermo Fisher; 1:1000). Then, blots were incubated with a peroxidase-coupled goat anti-rabbit secondary antiserum (32460; Thermo Fisher; 1:500) and the binding was visualized using SuperSignal™ West Femto Maximum Sensitivity Substrate (34095; Thermo Fisher) and a ChemiDoc™ MP Imaging System (BioRad). For band size determination PageRuler™ Plus Prestained Protein Ladder (10 to 250 kDa; 26620; Thermo Fisher) was used.