Rabbit anti cx3cr1 antibody

The rat brain tissue or cell samples were lysed in RIPA buffer containing phenylmethylsulfonyl uoride (PMSF) and a protease inhibitor cocktail as previously described. Lysates were centrifuged and collected.
Total protein concentration was determined using a BCA Protein Assay Kit (Beyotime). An equal amount of protein ranging from 15 to 30 µg total protein was separated by SDS-PAGE, blotted onto polyvinylidene di uoride (PVDF) membrane (Millipore). The following antibodies were used: rabbit anti-CX3CR1 antibody (1:3000, Abcam), goat anti-CX3CL1 antibody (1:200, Abcam), rabbit anti-BDNF antibody (1:3000, Abcam), rabbit anti-GAPDH antibody (1:3000, Service), goat anti-iba1 (1:500, Woko). Protein bands were visualized using a chemiluminescence system (ChemiDocTM XRS+, BioRad). The protein expressions were semi-quantitatively analyzed with ImageJ software.

Microglial cells were seeded on glass cover slips in 24-well plates. Cells were washed with PBS and cultured; the cultured cells were then fixed in 4% formaldehyde and permeabilized with 0.1% Triton X-100 for 5-10 min. Indirect immunofluorescence was performed using the following primary antibodies: rabbit anti-PU1 (1:200, Abcam, MA, USA; Ab88082), mouse anti-Ki67 (1:500, BD Pharmingen; 550609), mouse anti-F4/10 (1:200, Abcam; Ab6640), rabbit anti-CX3CR1 antibody (1:200, Abcam), rabbit anti-TMEM119 antibody (1:200, Abcam), rabbit anti-IBA-1 antibody
(1:500, Wako, MA, USA), and goat anti-IBA1 antibody (1:500, Abcam; Ab48004).
Cells were incubated with the primary antibodies diluted in 0.5% Triton X-100 in PBS containing 5% normal donkey serum at 4 °C overnight. After rinsing thrice with PBS for 5 min, Alexa 488-or Alexa-594-conjugated secondary antibodies (Abcam)
were used for detection. Nuclei were counterstained with 4′6-diamidino-2phenylindole (DAPI; Sigma). Cells without the addition of primary antibodies served as negative controls. Fluorescent images were taken using a confocal microscope (LSM 700, Carl Zeiss, Jena, Germany).