Aqueous hematoxylin

CD44v6/Myc double-staining was performed on 5-µm-thick paraffin-embedded xenograft sections. Antigen retrieval was performed using the PT link system (Dako, Agilent technologies, Santa Clara, CA, USA) with a 10 mM sodium citrate solution (pH 6.0). Before primary antibody incubation, we performed two consecutive blocking steps: one for 5 min with a 3% H₂O₂ solution to inhibit endogenous peroxidase and the other for 20 min with 10% human serum to reduce unspecific signals. Thereafter, sections were exposed to antibodies specific for CD44v6 (2F10, R&D systems, Minneapolis, MN, USA) and Myc (rabbit polyclonal, CST, Danvers, MA, USA), diluted in antibody diluent solution (Dako, Agilent technologies, Santa Clara, CA, USA). The MACH 2 Double Stain 2 kit (Biocare Medical, Pacheco, CA, USA) was used to reveal primary antibodies, using DAB and Vulcan Fast Red chromogens for detection. Aqueous hematoxylin (Sigma-Aldrich, St. Louis, MO, USA) was used to counterstain cell nuclei.
CR-CSphCs exposed to vemurafenib, trastuzumab and BKM120 were fixed, permeabilized and incubated overnight at 4 °C with CD44v6 and Myc antibodies. Then, cells were stained with Alexa Fluor-488 goat anti-rabbit IgG and Rhodamine Red-x goat anti-mouse IgG1 (Life Technologies, Waltham, MA, USA) secondary antibodies. Toto-3 Iodide (Life Technologies, Waltham, MA, USA) was used to counterstain nuclei.

Paraffin-embedded sections (5 μm-thick) of mouse lung tissues were exposed to antigen retrieval using heated retrieval solution (pH 6.0) and permeabilized for 10 min on ice with 0.1% Triton X-100 PBS. For immunohistochemistry analysis, sections were incubated overnight at 4 °C with anti-mitochondria antibody (ab92824, Abcam), anti-p27 antibody (#2552, Cell Signaling Technology) and anti-SOX9 antibody (Ab5535, Sigma–Aldrich) and subsequently exposed to biotin/streptavidin (LSAB 2 system-HRP, Agilent) and the 3-amino-9-ethyl carbazole (AEC, Agilent). Nuclei were stained with aqueous hematoxylin (Sigma–Aldrich). Images were obtained using a confocal microscope (Olympus BX60) and quantified with ImageJ - Color Inspector 3D.