Dead end fluorometric tunnel system

Cells were fixed in 4% polyformaldehyde (Sigma-Aldrich) for 15 min, and then subjected to TUNEL staining according to the manufacturer’s instructions (Dead End Fluorometric TUNNEL System, promega, G3250). DAPI was used to stain cell nuclei. Seven different fields per HPF were selected randomly to quantify the number of TUNEL-positive cells. TUNEL-positive cell nuclei were counted manually and compared with the total microglia number counted manually as DAPI and given as a percentage. The quantification data were from at least three independent experiments.

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.

This was performed using the DeadEnd™ Fluorometric TUNNEL system (Promega) following the manufacturer’s instructions and as described previously [39 (link)]. Briefly, the cells were fixed with 4% PFA, permeabilized with 0.2% (v/v) triton X-100, and incubated with the equilibration buffer containing the nucleotide mix and the rTdT Enzyme for 1 h at 37 °C. The reaction was terminated using the stop solution and the stained cells mounted on a glass slide using mounting media and visualized using a Nikon Eclipse 80i Microscope with an Etiga 2000R camera attached.