Tissue extraction reagent 1

The CC white matter tissues were dissected from the other half of the slices mentioned above in cold ACSF under an anatomic microscope. Then, the CC tissue was quickly transferred into Tissue Extraction Reagent 1 (FNN0071, Invitrogen, Camarillo, CA) with 1:1000 protease inhibitor (P-2714, Sigma) and homogenized. Protein concentration was measured by bicinchoninic acid assay (BCA assay). Routine electrophoresis was carried out using 10% sodium dodecyl sulfate-polyamide gel. Polyclonal rabbit anti-β-APP (1:600; AB5302, Millipore, Temecula, CA), monoclonal mouse anti-inducible nitric oxide synthase (iNOS; 1:500; AB49999, Abcam, Cambridge, MA), and rabbit anti-tumor necrosis factor alpha (TNF-α; 1:1000; AB66579, Abcam) were used to identify β-APP, iNOS, and TNF-α. Mouse anti-β-actin (1:10000; Sigma, A2228) was applied as a gel loading control. Immunoreactivity bands were detected using enhanced chemiluminescence and developed with autoradiography film.

The prepared brain slices were divided into the ACSF group and ACSF containing LPS 0.2 μg/mL group, both bathed for 3 hours or 6 hours. After an incubation that displayed significant β-APP accumulation, pre-treatment with RS-LPS (2.0 μg/mL) followed by incubation with LPS (0.2 μg/mL) or co-incubation with LPS+RS-LPS was performed for the same duration. Then, CC tissues were disseced and transferred to Tissue Extraction Reagent 1 (FNN0071, Invitrogen) with 1:1,000 protease inhibitor (P-2714, Sigma-Aldrich, St. Louis, MO, USA), and homogenized. Protein concentration was measured by bicinchoninic acid assay (BCA assay). Routine electrophoresis was carried out using 10% sodium dodecyl sulfate-polyamide gel. Polyclonal rabbit anti-β-APP antibody (1:600; #AB5302, Millipore, CA, USA) was used to detect β-APP. Mouse anti-β-actin (1:10,000, #A2228, Sigma-Aldrich) was applied as a gel loading control. Immunoreactivity bands were detected using enhanced chemiluminescence and developed with autoradiography film. Protein density of β-APP was measured using Image J and normalized to the corresponding β-actin in each sample.

The rats were euthanized with isoflurane and brain slices were prepared as the same way as described in the Electrophysiology section. The CC was dissected from coronal brain slices in cold ACSF under a dissecting microscope and quickly transferred into Tissue Extraction Reagent 1 (FNN0071, Invitrogen, Camarillo, CA) with 1:1000 protease inhibitor (P-2714, Sigma). Protein homogenate concentrations were determined by bicinchoninic acid assay (BCA assay). After centrifugation, the supernatant was subjected to sodium dodecyl sulfate-polyamide gel electrophoresis and then the protein was transferred to a PVDF membrane. The membranes were then blocked with 5% non-fat milk and incubated with rabbit anti-β-APP antibody (1:500; Abcam USA), followed by HRP-conjugated secondary antibody (1:5000; Jackson Immuno Inc., West Grove, PA). Mouse anti-β-actin (1:10,000; Sigma) was used for the gel loading control. Protein bands were detected using enhanced chemiluminescence and developed with autoradiography film. Image J (NIH, Bethesda, MD) software was used to quantify the band densities and protein densities of β-APP values were normalized by corresponding β-actin in each sample.