Rabbit anti mouse csf1r polyclonal antibodies

Primary neurons and MBEC4 cells were fixed with 4% paraformaldehyde for 10 min, permeabilized using 0.1% Triton X-100 for 5 min, and blocked using 5% normal goat serum in phosphate-buffered saline (PBS) for 1 h at room temperature. Neurons were incubated with rabbit anti-mouse IL-34 polyclonal antibodies (ProSci, Poway, CA, USA), mouse anti-mouse microtubule-associated protein–2 (MAP-2) monoclonal antibody (Chemicon, Temecula, CA, USA) overnight at 4°C followed by a 1-h incubation with Alexa-conjugated secondary antibodies (Invitrogen, Carlsbad, CA, USA). MBEC4 cells were stained using rabbit anti-mouse CSF1R polyclonal antibodies (Abcam, Cambridge, UK) overnight at 4°C followed by a 1-h incubation with Alexa-conjugated secondary antibodies (Invitrogen). Nuclei were counterstained with Hoechst 33342 (Invitrogen). Images were analyzed using a deconvolution fluorescent microscope system (BZ-8000, Keyence, Osaka, Japan).

Brains and lumbar spinal cords from C57BL/6J mice were fixed with 4% paraformaldehyde overnight, equilibrated in 20% sucrose with PBS for 48 hours, embedded in Tissue Tek O.C.T. compound (Sakura Finetechnical Co., Ltd., Tokyo, Japan), and frozen at −80°C overnight. Coronal brain sections and transverse spinal cord sections (20 µm-thick) were prepared using a cryostat. Sections were permeabilized using 0.3% Triton X-100 after blocking with 5% normal goat serum in PBS for 1 h. Sections were incubated with rabbit anti-mouse IL-34 polyclonal antibodies (ProSci), mouse anti-mouse MAP-2 monoclonal antibody (Chemicon), rabbit anti-mouse CSF1R polyclonal antibodies (Abcam), and Dylight 594–labeled tomato lectin (Vector Laboratories) overnight at 4°C followed by a 1-h incubation with Alexa-conjugated secondary antibodies (Invitrogen). Images were analyzed using a deconvolution fluorescent microscope system (BZ-8000, Keyence).