Toto 3 iodide

For fluorescent staining of Jagged-1, tissue sections were prepared as described for IHC, however antigen retrieval was accomplished by treatment with 20 μg/mL proteinase K diluted in TE, pH 9.0 for 20 min at 37 °C. Following incubation with primary antibody (rabbit anti-Jagged-1, Santa Cruz cat. #sc-8303; 1:100) and subsequent washes, tissues were incubated with a 1:500 dilution of secondary anti-rabbit Alexa-546 (Life Technologies cat. #A11010) for 2 h at room temperature (RT). Tissues were then washed 3 times for 10 min with 0.05% Tris buffered saline (TBS)-Tween, once for 5 min with TBS, and counterstained with TOTO-3 iodide (Life Technologies cat. #T3604; 1:2000) for 30 min at RT. Excess TOTO-3 was removed and slides were allowed to dry prior to mounting with Prolong® Gold anti-fade reagent (Life Technologies cat. #P10144).

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.