Each tumor sample was fixed in formalin and embedded in paraffin. The blocks were sliced into 5 μm-thick sections, which were deparaffinized in Histo-Clear (Cosmo Bio), hydrated in a graded series of alcohol, and subjected to heat-activated antigen retrieval. After blocking endogenous peroxidase activity, the tissue was incubated with CD44 monoclonal antibody (1 : 250, Cell Signaling Technology, number 3570S) for 4 hours at room temperature. Subsequently, the sections were washed and incubated with biotinylated secondary antibody for 30 minutes at room temperature. The reaction complexes were stained with diaminobenzidine and counterstained with hematoxylin.
For double-labeling immunofluorescence, sections were incubated with a mixture of two primary antibodies to CD44 (1 : 250, Cell Signaling Technology, number 3570S) and Nestin (1 : 250, EMD Millipore, ABD69) diluted in Tris-buffered saline containing 0.02% Tween 20 (TBST) and 0.1% bovine serum albumin in a humidified chamber overnight at 4°C. After washing with TBST, sections were treated with Cy3-conjugated anti-rabbit and DyLight 488-labeled anti-mouse IgG secondary antibodies (1 : 1000; Jackson ImmunoResearch, West Grove, PA). Hoechst 33342 (Sigma-Aldrich) was used for nuclear staining. The immunostained specimens were observed with a conventional microscope (BX52; Olympus, Tokyo, Japan).
For double-labeling immunofluorescence, sections were incubated with a mixture of two primary antibodies to CD44 (1 : 250, Cell Signaling Technology, number 3570S) and Nestin (1 : 250, EMD Millipore, ABD69) diluted in Tris-buffered saline containing 0.02% Tween 20 (TBST) and 0.1% bovine serum albumin in a humidified chamber overnight at 4°C. After washing with TBST, sections were treated with Cy3-conjugated anti-rabbit and DyLight 488-labeled anti-mouse IgG secondary antibodies (1 : 1000; Jackson ImmunoResearch, West Grove, PA). Hoechst 33342 (Sigma-Aldrich) was used for nuclear staining. The immunostained specimens were observed with a conventional microscope (BX52; Olympus, Tokyo, Japan).