For immunohistochemistry analyses, samples were fixed overnight in 4% formalin solution and embedded in paraffin. In brief, 3-µm-thick sections of FFPE tumors were deparaffinized and antigen retrieval was performed by boiling the sections in citrate buffer at pH 6.0 or EDTA at pH 9.0 for 20 min. Primary antibodies used were as follows: Dclk1 (1:50; pH 6.0; Abcam), Sox9 (1:50; pH 6.0; Millipore), Ki67 (1:50; pH 6.0; Cell Marque), corresponding secondary antibody detection kits for reduced background on murine tissue were used (Histofine Simple Stain Mouse MAX PO; Medac) and stained on an automated stainer (LabVision Autostainer 480S; Thermo Fisher Scientific). All slides were scanned with a Panoramic 250 slide scanner (3D Histech.com).
For immunofluorescence, frozen sections were incubated in the target retrieval solution (Dako) for 7 min. Primary antibodies are listed in the Cell lines and reagents section. Slides were analyzed using a sp5 confocal microscope (Leica).
For immunofluorescence, frozen sections were incubated in the target retrieval solution (Dako) for 7 min. Primary antibodies are listed in the Cell lines and reagents section. Slides were analyzed using a sp5 confocal microscope (Leica).