Ab183830

Immunofluorescence staining was performed according to our previously published protocols [41 (link), 42 (link)]. Briefly, the brain sections (6 μm thick) were incubated with primary antibodies against ALX/FPR2 (1:200, cat. no. ab203129, Abcam), GFAP (1:200, cat. no. MAB3402X, EMD Millipore, USA), Iba-1 (1:200, RRID: AB_2224402), and NeuN (1:200, cat. no. ABN78A4, EMD Millipore, USA) overnight at 4 °C. Then, the brain sections were incubated with the corresponding secondary antibodies conjugated with Alexa Fluor 488 and/or Alexa Fluor 594 (Jackson ImmunoResearch Incorporation, West Grove, PA, USA) for 1 h at room temperature, after which a ZEISS HB050 inverted microscope system was used for fluorescence detection.
For in vitro cell staining, primary antibodies against Iba-1 (1:200, RRID: AB_2224402) and p65 (1:200, 1:1000, cat. no. D14E12, CST) were used for microglia staining, while a primary antibody against MAP2 (1:500, cat. no. ab183830, Abcam) was used to stain neurons. The protocols and the secondary antibodies used for in vitro cell staining were the same as those used to stain brain sections.

MHY1485 (MCE, HY-B0795), Bafilomycin A1 (BafA1, MCE, HY-100558), anti-β-actin (Proteintech, 20536-1-AP), anti-p16 (SantaCruz, sc-1661), anti-p21 (SantaCruz, sc-6246), anti-γH2A.X (CST, 2577), anti-MAP2 (Abcam, ab183830), anti-MAP2 (Abcam, ab11268), mTOR (CST, 2983), p-mTOR (CST, 5536), LC3B (Affinity, AF4650), anti-p62 (CST, 23214), anti-ATG7 (Proteintech, 67341-1-lg), anti-REST (Novus, NBP1-82399).

Immunostaining was performed in primary cortical neurons and NE-4C cells. Cells seeded on coverslips were fixed with cold methanol (≥99.8%, yongda chemical reagent, Tianjin) for 10 min, and then incubated in 5% Bovine serum albumin (BSA, >99%, Solarbio) for 2 h to block nonspecific binding of IgG. Then the cells were reacted with primary antibody at 4°C overnight. The primary antibodies used in this experiment were rabbit monoclonal antibody against H₃R (ab124732, Abcam, 1:200), mouse monoclonal antibody against Nestin (ab11306, Abcam, 1:200) and rabbit monoclonal antibody against MAP-2 (ab183830, Abcam, 1:100). After repeated washing in phosphate-buffered saline (PBS), cells were incubated with secondary antibody in 3% BSA for 2 h at 25°C. The secondary antibodies used in this experiment were donkey anti-rabbit IgG-Alexa Fluor 488 (A21206, Invitrogen, 1:500) and donkey anti-mouse IgG-Alexa Fluor 546 (A10036, Invitrogen, 1:500). After further washing in PBS, cultures were dried, and mounted on glass slides. The stained cells were observed under a laser scanning confocal microscope (Leica TCS SPE, Germany). Total dendritic length and neuronal complexity were quantified by using ImageJ software and the Fiji plugins Simple Neurite Tracer Analysis as well as Sholl Analysis.