Elimination of leukemic infiltration associated with decreased survivin expression and induction of apoptosis in spleens of leukemic rats responsive to nanoliposomal C₆ ceramide. Images were taken from hematoxylin and eosin or immunohistochemically stained 5-μm paraffin sections of rat spleen tissues and analyzed with Olympus 1 × 51 Microscope (Olympus America) attached to Spot Insight Camera (Diagnostic Instruments). The software for imaging and analyses was SPOT Advanced Plus Imaging software. (A) Spleen sections from rats treated with ghost nanoliposome (Ai); with C₆ ceramide nanoliposome, responsive (Aii), or nonresponsive (Aiii); and from normal control rat (Aiv) were stained with hematoxylin and eosin. (B) Spleen sections from rats treated with ghost nanoliposome (Bi); from C₆-ceramide nanoliposome responsive (Bii), or nonresponsive (Biii); and from normal control rat (Biv) were stained with anti-survivin antibody (D-8; Santa Cruz Biotechnology). (C) Spleen sections were stained with an In Situ Cell Death Detection kit to assess the degree of cellular apoptosis via TUNEL-TMR red staining (red); 4′,6-diamidino-2-phenylindole-stained nuclei (blue). Spleen sections from rats responsive to nanoliposomal-C₆ (Cii) showed more positive TUNEL staining than sections obtained from rats treated with ghost nanoliposome (Ci), from rats not responding to C₆-ceramide nanoliposome (Ciii) and from normal control rat (Civ).