488 conjugated secondary antibodies

Cells were seeded onto collagen I-coated coverslips. Following transfection or incubation overnight, cells were fixed in 4% paraformaldehyde in PBS for 20 min at RT and subsequently permeabilised with 0.2% Triton X-100 in PBS for 5 min. For F-actin staining, cells were incubated with either TRITC- or Alexa fluor 488-conjugated phalloidin (Invitrogen) diluted in PBS for 1 h at RT. Following this incubation, cells were washed 3 times in PBS. For detection of paxillin, primary antibodies were diluted in PBS with 3% bovine serum albumin (Fisher Scientific) and incubated for 2 h at RT. Following labeling with the primary antibody, cells were washed 3 times with PBS before incubation with either Alexa fluor 568 or 488-conjugated secondary antibodies (Invitrogen) and phalloidin. Cells were then imaged using an Olympus IX71 microscope with a 40X/NA 1.3 UPlanFLN oil-immersion objective and Image-Pro Plus software (supplied by MAG, UK).

For tissue samples, the paraffin-embedded tissue sections were deparaffinized and rehydrated. Slides were boiled in 10 mM citrate buffer (pH 6.0) for 40 min for the antigen retrieval. For cells staining, cells were fixed with 4% paraformaldehyde for 10 min at room temperature, and washed thrice with PBS. Nonspecific binding was blocked with 5% bovine serum albumin for 30 min. The slides were incubated with the primary antibodies against complement component 9 (C9, Abcam), HLA-DR (Abcam), CD163 (Abcam), HIF1α (Cell Signaling Technology), and CD68 (Abcam) at 4 °C overnight, followed by incubation with Alexa Fluor® 594 or 488-conjugated secondary antibodies (Invitrogen). For confocal microscopy, the cells on coverslips were counterstained with DAPI (Life Technology) and imaged using a confocal laser-scanning microscope (Olympus FV1000).

Two mice in each group were deeply anesthetized with avertin and perfused with PBS (pH 7.4) and 4% (wt/vol) paraformaldehyde in PBS. Brains were collected, post-fixed, and sectioned at 40 μm thickness using a vibratome (Leica VT1200, Leica Biosystems, Nussloch, Germany). Brain sections were incubated at 4 °C overnight with antibodies for ΔFOSB (1:200, 2251S, Cell Signaling; stains, both FOSB and ΔFOSB but mainly ΔFOSB, because FOSB degrades with time, leaving ΔFOSB), glial fibrillary acidic protein (GFAP; 1:200, Z0334, Dako, Glostrup, Denmark), glutamine synthetase (GS; 1:200, MAB302, Millipore, Temecula, CA, USA), ionized-calcium-binding adaptor molecule 1 (IBA1; 1:200, ab5076, Abcam, Cambridge, UK), sodium-dependent neutral amino acid transporter 2 (SNAT2; 1:20, sc-166366, Santa Cruz, Dallas, TX, USA), phosphate-activated glutaminase (PAG; 1:200, AB113509, Abcam), insulin receptor β (IRβ; 1:20, sc-57342, Santa Cruz), insulin receptor substrate-1 (IRS-1; 1:200, 06-248, Millipore), vesicular glutamate transporter 1 (VGLUT1, 1:20, 48-2400, Invitrogen, Carlsbad, CA, USA), and VGLUT2 (1:20, 42-7800, Invitrogen). The slices were then incubated with Alexa Fluor 594- and/or 488-conjugated secondary antibodies (1:200, Invitrogen). Digital images were captured using a spinning-disk confocal microscope (Olympus, Tokyo, Japan) and analyzed with ImageJ software (NIH).

The mice were anesthetized with avertin (0.25 g/kg via intraperitoneal injection) and perfused with PBS and neutrally buffered in 4% paraformaldehyde as previously described [14 (link)]. Brains were removed, postfixed over 6 h, and sectioned at a thickness of 40 μm. The sections were incubated with the following antibodies for 1–2 days at 4 °C: anti-Aβ_1–16 (6E10) (#803015, Biolegend, San Diego, CA, USA, 1:1000), anti-iNOS (sc-7271, Santa Cruz Biotechnology, Dallas, TX, USA, 1:20), and anti-ionized calcium-binding adapter molecule 1 (IBA-1) (ab5076, Abcam, Cambridge, UK, 1:200). The sections were washed and then incubated with Alexa Fluor 594- and/or 488-conjugated secondary antibodies (Invitrogen, 1:1000). The binding specificity of the primary antibodies was confirmed with secondary antibody-only controls. Digital images were obtained using a confocal microscope equipped with an Olympus Disk Spinning Unit (Olympus, Tokyo, Japan). Fluorescence intensity or signal-positive cell numbers were analyzed using ImageJ software (NIH, RRID:SCR_003070).