4.4. Immunofluorescence Staining of Mouse Testicular Tissue

Immunofluorescence staining of 4µm thick sections from formalin-fixed, paraffin-embedded testicular tissue blocks was performed as described by Huleihel et al., 2013 [33]. Polyclonal rabbit anti-mouse IL-34 antibodies (USBI, United States Biological-141047, Salem, MA, USA) (0.2 mg/mL; 1:40; final dilution) or polyclonal rabbit anti-mouse CSF-1R antibodies (LS-C164350, LifeSpan BioSciences, Seattle, WA, USA) (1:200 final dilution) were used as primary antibodies. The fluorescence antibody (Cy-3; donkey anti-rabbit antibodies; 1:1000; Jackson ImmunoResearch, West Grove, PA, USA) were applied as secondary antibodies. After overnight incubation at 4 °C, the antibodies were washed by PBS and DAPI staining and Molecular Probes (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) were added. Negative controls were included for each specimen using PBS/casein/FCS/BSA or relevant IgG isotype instead of the primary antibodies.