Anti phalloidin

Rabbit anti-mouse lyve-1 antibody (RDI-103PA50) was from Research Diagnostics Incorporated (Concord, MA). Donkey anti-rabbit and rat IgGs conjugated with alexa 488, 594 were from TebuBio (TebuBio, Le Perray en Yvelines, France). Anti-pSer473Akt, anti-Akt, anti-pErk, anti-Erk1/2 are from Cell Signalling Technology. Anti-Phalloidin was from Cytoskeleton (actin-stain phalloidin 488, cat PHDG1). Anti-phospho-VEGFR-3 is from Cell applications Inc. Rabbit anti-human LC3B, anti-human Notch/NCID and anti-p62 were from Cell Signaling (#2775), Goat anti-human VE-Cadherin from Santa-Cruz ((C19):SC6458) and Rabbit anti-human ATG5 and ATG7 from Sigma. Anti-human active beta-catenin was from Milliopore.

The use of fresh breast tumor specimens was approved by the research ethic committees at the Korea National Cancer Center (NCC). Informed consent was obtained from all patients. All experiments with human specimens were performed in accordance with relevant guidelines and regulations of NCC. Samples were fixed with 4% paraformaldehyde for fluorescent staining. Samples were permeabilized with 0.3 M glycine and 0.3% Triton X-100, and nonspecific binding was blocked with 2% normal swine serum (DAKO, Glostrup, Denmark). Staining was performed as described previously56 (link), using the primary anti-Wnt1 (Abcam), anti-Phalloidin (Cytoskeleton Inc.), anti-ALDH1 (Abcam), anti-TCF41 (Abcam), anti-PCNA (Abcam), and anti-LEF1 (Cell Signaling Technology). Samples were examined by fluorescence microscopy (Zeiss LSM 510 Meta). The calculation of expression was based on green fluorescence area and density divided by cell number, as determined from the number of DAPI-stained nuclei, in three randomly selected fields for each specimen from a total of three independent experiments. For quantitation, an arbitrary threshold was set to distinguish specific from background staining, and this same threshold setting was applied to all the samples analyzed.