Anti csf1r

Phospho-CSF1R (Tyr723) (49C10) Rabbit mAb (RRID: AB_2085229), β-catenin (6B3) Rabbit mAb (RRID: AB_331149), anti-Phospho-Akt (Ser473) (RRID: AB_329825) and anti-total-Akt antibodies (RRID: AB_329827) were all bought from Cell Signaling Technology. Anti-GAPDH (RRID: AB_2630358), anti-CSF1R (RRID: AB_2085251), anti-Aβ (MOAB2), anti-β-actin antibodies (RRID: AB_306371) and Alexa Fluor 647 Donkey Anti-Rat IgG H&L (RRID: AB_2813835), as well as the BrdU Cell proliferation ELISA Kit, were all from Abcam. Rat Anti-mouse CD68 monoclonal antibody (RRID: AB_322219) was from Bio-Rad. Anti-HA-tag (RRID: AB_11042321) antibody and anti-Myc-tag antibody (RRID: AB_11182162) were from Proteintech. Rabbit anti-Iba1 (RRID: AB_2687911) was from Wako. Alexa Fluor 488 Goat anti-Rabbit (RRID: AB_10374301), Alexa Fluor 594 Goat anti-Rabbit (RRID: AB_10374440), Alexa Fluor 647 Goat anti-Rabbit antibodies (RRIDAB_10371940) and TRIzol reagent were all from Thermo Fisher Scientific. Granulocyte-macrophage colony stimulating factor (GM-CSF) and CSF1 were from R&D Systems. Basic Glial Cells Nucleofector Kit was purchased from LONZA. ReverTra Ace qPCR RT Master Mix was from TOYOBO. FastStart Universal SYBR Green Master mix was from Roche. CellTiter 96^R Aqueous One Solution (MTS assay) and DeadEnd™ Fluorometric TUNNEL System were from Promega.

To detect the DC-SIGN⁺ and CD169⁺ macrophages derived from THP-1 cells induced by CSF-1 and blocked with CSF-1R, we conducted a cellular immunofluorescence analysis. We treated the differentiated macrophages with CSF-1 (50 ng/ml) and cultured them for 48 h. For confirmation, we cultured the differentiated macrophages with RPMI 1640 medium (Gibco, China) containing anti-CSF-1R (Abcam, UK, 1:20) for 2 h and then added CSF-1 (R&D Systems, USA, 50 ng/ml) to the medium for 48 h. Cellular immunofluorescence staining was performed as previously described [34 (link)]. The target macrophages were washed twice with PBS, fixed with 4% paraformaldehyde (pH 7.0) for 30 min, permeabilized with 0.1% Triton X-100 (Sigma) for 10 min, blocked with 10% normal goat serum for 1 h, and incubated overnight with rabbit anti-human CD169 (ab183356, Abcam, UK; 1:100) or anti-DC-SIGN (ab5715, Abcam, UK; 1:50) antibodies and a mouse monoclonal antibody against human CD68 (ab955, Abcam, UK; 1:200) at 4 °C. The cells were then washed with PBS three times and incubated at room temperature with a DyLight 488-conjugated donkey anti-rabbit secondary antibody (1:400; Abcam, UK) or a DyLight 594-conjugated donkey anti-goat secondary antibody (1:400; Abcam, UK) for 1 h. After the plates were washed, cell nuclei were counterstained with fluorescent mounting medium with 4’,6-diamidino-2-phenylindole (Abcam, UK).