Anti iba 1

Protein lysates were extracted according to a previously described method (Hwang et al. 2018 ; Kim et al. 2018 (link)). Dissected hippocampal tissues were homogenized in 400 μl per 1 g concentration of RIPA buffer (Cell Signaling Technology, Beverly, MA, USA) with 1 mM PMSF (Sigma-Aldrich Inc.) on ice. The homogenized sample was incubated on ice for 20 min. Incubated sample was centrifuged at 14,000 g for 10 min at 4°C, and supernatants were collected. Protein content was measured using a micro-drop plate reader (Thermo Fisher Scientific). NF-кB and IкBα in the hippocampus were detected using nuclear/cytosol fractionation kit (BioVision Inc, Milpitas, CA, USA) according to the manufacturer’s instructions. The following primary antibodies (1:1000 dilution) were selected to react overnight at 4°C: mouse anti-β-actin, anti-TNF-α, anti-interleukin-6 (IL-6), anti-proBDNF, anti-BDNF, anti-Iba-1, rabbit anti-TrkB, anti-NF-κB, and anti-IκBα (Santa Cruz Biotechnology). Subsequently, membranes were incubated for 1 h with attempt secondary antibodies (1:2000; Vector Laboratories). Blot membrane was detected using the HRP-conjugated IgG (Vector Laboratories) and the enhanced chemiluminescence detection kit (Bio-Rad, Hercules, CA, USA). Detected bands were quantified by Image-Pro® plus image analysis system (ver. 6.0, Media Cybernetics Inc., Silver Spring, MD, USA).

Alpha-linoleic acid (cis-9,cis-12-octadecadienoic acid, Lot#30H8479) and Aβ_1–42 peptides were purchased from Sigma-Aldrich Chemical Co., St. Louis, MO, USA. The antibodies used in the study included anti-Aβ (sc-28365), anti-p-Tau (sc-390476), anti-BACE-1 (sc-33711), p-Tau (sc-)anti-PSD-95 (sc-71933), anti-synaptosomal-associated protein 23 (SNAP-23) (sc-374215), anti-p-JNK (sc-6254), anti-Caspase 3 (sc-7272), anti-Bax (sc-7480), anti-Bcl-2 (sc-7382), anti-PARP-1 (sc-7008), anti-TNF-α (sc-52746), anti-p-NF-κB p65 (Ser536) (sc-136548), anti-TLR4 (sc-293072) anti-Iba-1 (sc-32725), anti-GFAP (sc-33673), and anti-β-actin (sc-47778) from Santa Cruz Biotechnology (Dallas, TX, USA). The primary antibodies were diluted in TBST (1:1000) (Santa Cruz Biotechnology) and the secondary anti-mouse HRP conjugated (Promega Ref# W402) and antirabbit HRP conjugated (Promega Ref# W401) antibodies that were diluted to 1:10,000 in 1 × TBST were obtained from Promega, USA. For confocal microscopic studies, the secondary fluorescent antibodies used were goat anti-mouse (Ref# A11029) and goat anti-rabbit (Ref# 2732), which were diluted in 1× phosphate-buffered saline (PBS).

The immunohistochemical techniques used have been previously described [52 , 58 (link), 60 (link)]. Slices were incubated overnight with one of the following primary antibodies (specific for each whether polyclonal or monoclonal): anti-TH (Millipore, 1:500 in PBS, v/v), anti-DAT (Santa Cruz Biotechnology, 1:300 in PBS, v/v), anti-α-syn (Santa Cruz Biotechnology, 1:50 in PBS, v/v), anti-Iba-1 (Santa Cruz Biotechnology, 1:300 in PBS, v/v), and anti-GFAP (Santa Cruz Biotechnology; 1:200 in PBS, v/v). Immunohistochemical images were collected using Leica DM6 (Milan, Italy) associated with an Imaging system (LasX Navigator, Milan, Italy). The digital images were opened in ImageJ, followed by IHC profiler plug-in. All immunohistochemical analyses were carried out by two observers blinded to the treatment [29 (link), 54 (link), 61 –63 (link)].

Anti-MMP-12, anti-MMP-9, anti-GFAP, anti-MOG, anti-Iba1, anti-TNFα, anti-TNFR1, anti-TNFR2, anti-caspase-3, anti-cytochrome C, anti-AIF, and anti-MBP antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-NeuN antibody was obtained from Millipore (Billerica, MA). Anti-glial fibrillary acidic protein (GFAP) was obtained from Dakocytomation (Carpinteria, CA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody was obtained from Novus Biologicals (Littleton, CO).

Tissue samples from the brain were processed in brief the expression of Glial fibrillary acidic protein (GFAP) and IBA1 was quantified in cytosolic fraction from brain tissues. The filters were blocked with 1 × PBS, 5% (w/v) non fat dried milk (PM) for 1 h at room temperature and then probed with specific Abs anti-GFAP (1/500; Santa Cruz Biotechnology) or anti-IBA1 (1/500; Santa Cruz Biotechnology), in 1 × PBS, 5% w/v non fat dried milk, 0.1% Tween-20 (PMT) at 4°C, overnight. Signals were detected with enhanced chemiluminescence (ECL) detection system reagent and the relative expression of the protein bands was quantified by densitometry with BIORAD ChemiDoc^TMXRS+software, standardized to β-actin and lamin A/C levels, as previously described [10 (link)].

Paraffin sections were deparaffinied with xylene and rehydrated by gradient of ethyl alcohol from 100 to 70%. Sections were washed with PBS and reacted with normal goat serum for 1 h for blocking non-specific reaction. Sections were incubated in a wet chamber for overnight at 4 °C with following primary antibodies: anti-Iba-1, anti-GFAP, anti-NF-κB, anti-TNF-α, and anti-IL-1β (anti-Iba-1, anti-GFAP, anti-NF-κB, anti-TNF-α, and anti-IL-1β primary antibody, diluted 1:100, Santa Cruz Biotechnology). Sections were washed with PBS and incubated with fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG or anti-rabbit IgG (1:200, Santa Cruz Biotechnology) for 2 h at room temperature. Sections were reacted with 4′,6-diamidino-2-phenylindole (DAPI, Sigma Aldrich) for 10 min and mounted with Ultra-Cruz mounting medium (Santa Cruz Biotechnology). Fluorescent signals were observed and imaged with a confocal microscope (FV-1000, Olympus, Tokyo, Japan). Integrated intensities of positive signals were analyzed by Image-Pro Plus image software (Media Cybernetics, Rockville, MD, USA). Intensity values were expressed as a ratio of LPS-treated group intensity to control group intensity. Intensity value of control group was set to 1.

Lipopolysaccharide and caffeine were purchased from Sigma-Aldrich Chemicals Company (St. Louis, MO, USA). The antibodies used in the Western blot and immunofluorescence studies were anti-Nrf2 (sc-722), anti-HO1 (sc-136,961), anti-synaptosomal-associated protein 23 (SNAP-23) sc-374,215), anti-PSD-95(sc-71,933), anti-TNF-α (sc-52,746), TLR4 (sc-16240), anti-Bax(sc-7480), anti-Bcl2 (sc-7382), anti-p-NF-kB (sc-36,548), anti-Iba-1 (sc-32,725), anti-Glial fibrillary acidic protein (GFAP; sc-33,673), and anti-β-actin (sc-7,778) (Santa Cruz Biotechnology, Dallas, TX, USA). In addition, the anti-Cleaved Caspase-3 #9664) antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). Other primary antibodies were diluted in 1× TBST (1:1000), and secondary anti-mouse horseradish peroxidase (HRP)-conjugated (Promega Ref#W402) and anti-rabbit HRP-conjugated (Promega Ref# W401) antibodies were diluted 1:10,000 in 1× TBST(Sigma, Fitchburg, WI, USA). For the confocal microscopic studies, secondary fluorescent antibodies goat anti-mouse (Ref# A11029) and goat anti-rabbit (Ref# 32732) diluted in 1× PBS were used.