Figure 5: DDA treatment induced in vivo differentiation of B16F10 and TS/A tumours implanted into immunocompetent mice.

Histology of tumours resected from immunocompetent mice treated with vehicle (left panels) or DDA (3.7 μg kg⁻¹) (right panels) and stained for specific markers of cell differentiation. Paraffin sections were stained with haematoxylin and eosin for histomorphological analyses. (a) B16F10 tumour sections from mice treated for 23 days were analysed for melanin production with Masson–Fontana staining (black labelling). (b) TS/A tumour sections from mice treated for 30 days were analysed for MFGE8 expression with a specific antibody (brown staining, indicated by arrows) and for neutral lipid production by ORO staining (red labelling, indicated by arrows). For lipid staining, frozen sections were fixed in 10% neutral-buffered formalin and then stained with ORO and counterstained with haematoxylin. Immunohistochemical staining was done on paraffin-embedded tissue sections, using a specific hamster monoclonal to MFGE8 antibody (4 μg ml⁻¹). Immunostaining of paraffin sections was preceded by an antigen retrieval technique by heating in 10 mM citrate buffer, pH 6, with a microwave oven twice for 10 min each. After incubation with the antibody for 1 h at room temperature, sections were incubated with biotin-conjugated polyclonal anti-hamster immunoglobulin antibody followed by the streptavidin-biotin-peroxidase complex (Vectastain ABC kit, Vector Laboratories, CA) and were then counterstained with haematoxylin. Negative controls were incubated in buffered solution without primary antibody. Magnification is × 200.