Anti app

Poly (lactide-co-glycolide) (PLGA)–PEG, sodium deoxycholate, Aβ_1–42 peptide and dichloromethane (DCM) were purchased from Sigma. All other chemicals used were of analytical grades. The anti-Bax, anti-Bcl-2, anti-APP, anti-BACE-1, anti-Nrf2, anti-HO-1, anti-p-NF-kB 65, anti-p-JNK, anti-TNFα, anti-iNOS, anti-p-P38, antibodies were purchased from Santa Cruz Biotechnology. Anti-caspase-3 and anti-actin antibodies were bought from Cell Signaling and anti-8-oxoguanine (anti-8-Oxo-G) were purchased from (Millipore, Billerica, MA, USA). The secondary antibodies used in our experiments were goat anti-mouse IgG, goat anti-rabbit IgG and rabbit anti-goat IgG, purchased from Santa Cruz Biotechnology.

Antibodies used include anti-STEP₆₁ (catalogue SC-23892, Santa Cruz), anti-GluN2B (#14544, Cell Signaling), anti-ERK1/2 (SC-154, Santa Cruz), anti-GluA2 (#5306, Cell Signaling), anti-APP (#SC-28365, Santa Cruz), and anti-β-actin (#4967, Cell Signaling). Phosphorylation site specific antibodies used include anti-GluN2B-pTyr¹⁴⁷² which recognizes phosphorylated Tyr-1472 of GluN2B (P1516-1472, PhosphoSolutions), anti-ERK1/2-pThr²⁰²/Tyr²⁰⁴ which recognizes phosphorylated Thr²⁰²/Tyr²⁰⁴ of ERK1 and Thr¹⁸⁵/Tyr¹⁸⁷ of ERK2 (#9106, Cell Signaling), anti-GluA2-p3Y which recognizes phosphorylated Tyr⁸⁶⁹, Tyr⁸⁷³, and Tyr⁸⁷⁶ (3Y) of GluA2 (#3921S, Cell Signaling), and anti-GluA2-pY⁸⁷⁶ which recognizes phosphorylated Tyr⁸⁷⁶ of GluA2 (#4027S, Cell Signaling).

Western blots were performed on whole brain and hippocampus as already described [50 (link),51 (link)]. Specific primary antibodies, i.e., anti-ikb-α (Santa Cruz Biotech, sc-1643), or anti-NFkB (Santa Cruz Biotechnology, sc-8008), or anti-Nrf2 (Santa Cruz Biotechnology, sc-36594), or anti-HO-1 (Santa Cruz Biotechnology, sc-136970), or anti-p-Tau (Santa Cruz Biotechnology, sc-32275). or anti-APP (Santa Cruz Biotechnology, sc-32277) were mixed in a 5% w/v nonfat dried milk solution and were incubated at 4 °C, overnight. Afterwards, blots were incubated with a peroxidase-conjugated bovine anti-mouse IgG secondary antibody or a peroxidase-conjugated goat antirabbit IgG (Jackson Immuno Research) for 1 h at room temperature [52 (link),53 (link)]. To verify the amounts of protein were equal, membranes were also incubated with an antibody against β-actin (Santa Cruz Biotechnology). Signals were detected with an enhanced chemiluminescence detection system reagent (Super-Signal West Pico Chemiluminescent Substrate, Pierce) [54 (link),55 (link)]. The relative expression of the protein bands was quantified by densitometry with Bio-Rad ChemiDoc XRS software and standardized to β-actin levels [56 (link)]. Images of blot signals were imported to an analysis software (Image Quant TL, v2003).

For immunofluorescence the brains (n=5 per group) were embedded in paraffin as previously described [7 (link)] and coronally sectioned (5μm) using a microtome. Brain sections including the cerebral cortex and the hippocampus were mounted on slides and deparaffinized in xylene solution. Then, the slides were hydrated in a series of graded ethanol (96%, 85%, 70%, 50%) for 5 minutes each. After washing in water and PBS the slides were incubated with 3% BSA/PBS for 1 h. Next, the sections were incubated with anti-APP (1:50) (Santa Cruz) at 4°C overnight. After washing in PBS, the samples were incubated with anti-rabbit Cy3-conjugate secondary antibody (1:500; SIGMA). For nuclear staining, the sections were incubated with Hoechst 33258 (5μg/ml) for 20 minutes. After washing in PBS the slides were mounted with cover slips and images were visualized by using a Leica DM5000 upright microscope (Leica Microsystems, Heidelberg, Germany) at 20X magnification.

Immunohistochemistry was performed as previously described (Lee et al., 2019 (link)). To detect target proteins, protein-specific primary antibodies were purchased from Novus Biologicals (anti-APP), Santa Cruz Biotechnology (anti-GFAP and anti-β-actin), Cell Signaling Technology (anti-iNOS and anti-COX-2), and Abcam (anti-BACE1, anti-IBA-1, and anti-Foxp3). Secondary antibodies were purchased from Vector Laboratories (anti-rabbit; Burlingame, CA, USA) and Santa Cruz Biotechnology (anti-goat).