Detection of melanocyte-specific HDGF-overexpression. A: Immunofluorescence of skin samples from wildtype, HDGFTyr, HDGFTyr/HDGF-/- and HDGF-/- mice. Paraffin sections were stained with affinity-purified anti-mouse HDGF antibody. Bound antibody was detected by incubation with Cy3 conjugated secondary anti-rabbit antibody. Nuclei were stained with DAPI. a-e HDGF; f-j merge of HDGF, DAPI and transmitted light. Green arrows point at HDGF positive melanocyte nuclei. Scale bar equates 100 μm. B: Primary melanocyte cell culture. Melanocytes were isolated from newborn HDGFTyr, wildtype and HDGF-/- mice. After 2-3 weeks melanocytes from wildtype and HDGF-/- mice grew in a sub-confluent cell layer, whereas very few melanocytes from HDGFTyr animals (red arrows) were able to survive in cell culture. a-c 100 × magnification; d-f 400 × magnification. Scale bar equates 100 μm. C: Western blot analysis of melanocyte lysates. a HDGF-level of primary melanocytes from HDGFTyr, wildtype and HDGF-/- mice was compared to the level of complete skin and B16F10 (murine melanoma cell line) lysates. 10 μg total protein was loaded onto an SDS-gel. b Equal loading of melanocyte lysate was assessed by staining the membrane with Coomassie solution.