Pa5 79481

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.

Formalin-fixed tissues were embedded in paraffin and sectioned by the Histology Core. Boiling citrate buffer was used for antigen retrieval, and endogenous peroxidase activity was quenched using hydrogen peroxide. Tissue sections were stained with antibodies against IL-18 (1 μg/mL, PA5-79481, Thermo Fisher Scientific), and Col6a3 (20 µg/mL, PA5-49914, Thermo Fisher Scientific), as described [34 (link)]. Sections were then labeled with biotinylated secondary antibodies (anti-rabbit, Cell Signaling Technology, Danvers, MA, USA; anti-rat, Vector Labs, Burlingame, CA, USA), as previously described. [34 (link)] A DAB substrate (Cell Signaling) was used for signal detection, as per manufacturer-provided protocols, and sections were counterstained with hematoxylin (Vector Labs). The Fiji ImageJ image processing package (version ImageJ2) was used for quantification of intensity of DAB staining by the color deconvolution method [53 (link)] and mean gray value was used to calculate optical density by the formula OD = log (max intensity/mean intensity, with a maximum intensity of 255 for 8–bit images [54 (link)].