Abi perkin elmer prism 5700 sequence detection system

Mice were euthanized and brains perfused and removed as above, after which they were either lysed using 1 ml of protein lysis buffer (50 mM NaCl, 20 mM HEPES, 1 mM EDTA, 2% Triton-X 100, 10% glycerol, with proteinase and phosphatase inhibitor) to extract total protein or 1 ml of TRIZol solution to extract RNA. NF-κB, Ikb-α, APP, beta secretase 1 (BACE-1), and presenilin 1 (PS1) levels were detected using western blot (4–12% Nupage Bis-Tris gel, Thermo Fisher Scientific) using standard technique (antibody: #8242S, #9242S, 1:1000, Cell signaling, Danvers, MA; ab 32136, 1:5000, Abcam; ab 2077, 1:1000, Abcam; MAB 5232, 1:1000, Millipore Sigma, Burlington, MA). Uncropped western blots in main figures are shown in Supplementary Figure 7. Relative expression of mRNA for APP was detected by two-step, real-time quantitative reverse transcription-PCR (RT-PCR) with the ABI Perkin Elmer Prism 5700 Sequence Detection System (Applied Biosystems, Foster City, CA) using Taqman probe (Mm00476361, Mm01344172, Invitrogen, Carlsbad, CA).

Total RNA was extracted using TRIzol from the right post-caval lobe (15596026, Thermofisher scientific, Waltham MA). Relative expression of mRNA for MUC5AC, MMP9, MMP12 and ELANE were detected by two-step, real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) with the ABI Perkin Elmer Prism 5700 Sequence Detection System (Applied Biosystems, Foster City, CA) using Taqman probe (Mm01276718, Mm00442991, Mm00500554, Mm00469310, Thermofisher scientific, Waltham MA). All data were normalized to eukaryotic 18s rRNA endogenous control (thermofisher scientific, Waltham, MA).