Immunocytochemistry.

For fluorescent microscopy, cryostat sections (5 µm) were fixed in acetone, washed in PBS, and blocked for 1 h at room temperature with 10% normal goat serum and 0.5% bovine serum albumin in PBS (blocking buffer). Slides were washed and incubated for 1 h with optimally diluted primary antibodies (anti-Bu-1 [clone AV-20; AbD Serotec], anti-CD8α [clone 3-298, AbD Serotec], and anti-CD8β [clone EP42, AbD Serotec]) and anti-CSF1R (55) or isotype controls, all diluted in blocking buffer. Sections were washed, incubated with an Alexa Fluor 488-labeled goat anti-mouse IgG1/IgG2a or Alexa Fluor 568-labeled goat anti-mouse IgG1/IgG2b according to the appropriate isotype, and diluted in blocking buffer for 1 h. Nuclei were visualized using DAPI (4′,6′-diamidino-2-phenylindole (Invitrogen). Images were captured with a Leica DMLB fluorescence microscope with a coupled device digital camera and analyzed using ImageJ analysis software. For light microscopy, cryostat sections (5 µm) were fixed in acetone and stained with Harris’ hematoxylin (Sigma-Aldrich) and 1% eosin (Sigma-Aldrich). Sections were dehydrated through graded ethanols and xylene and mounted in a xylene-based medium (DePex; Gurr-BDH Chemicals). Images were captured with a Hamamatsu Nano-zoomer-XR digital slide scanner.