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com/FedericoV/FractalMammaryLobule and instructions on how to use it can be found in the Supplemental Experimental Procedures. Processing of Breast Tissue Samples Normal breast tissue was obtained with informed consent from patients undergoing mammoplasty for aesthetic or prophylactic reasons, under protocols approved by Guy��s Research Ethics Committee in agreement with the Human Tissue Act. The tissue was processed as previously described (Ginestier et?al., 2007). Pieces of tissue were TRIB1 fixed in formalin for 24�C48?hr before being processed and embedded in paraffin. Immunostainings Paraffin-embedded sections (3�C4?��m) of normal human breast epithelium were deparaffinized in xylene and rehydrated in graded alcohol. Antigen retrieval was achieved by heating slides in pH 6 or pH 9 buffer (Vector Laboratories) according to recommendations or by enzymatic digestion with trypsin. Single and double IHC was performed with EnVision HRP Rabbit/Mouse (DAB+) and EnVision G2 Doublestain System Rabbit/Mouse (both from Dako), respectively, according to recommendations, except for ALDH1A3 that was detected using peroxidase-conjugated donkey anti-goat secondary antibody (Jackson Laboratory) followed by VECTASTAIN Elite ABC Kit (Vector Laboratories) and 3,3-diaminobenzidene (DAB). Primary antibodies used are summarized in the Supplemental Experimental Procedures. For double and triple IF stainings, sections were blocked with 10% donkey serum for 1?hr. Staining with primary antibodies was done in 10% donkey serum overnight at 4��C. Secondary antibodies (conjugated with AlexaFluor-488, -555, or -647, Molecular Probes) were incubated in 10% donkey serum for 1?hr at room temperature. Nuclei this website were counterstained with DAPI. All incubations and washes were done in PBS with 0.1% Triton X-100. Lobule Classification and Analysis of ALDH1A1 Representation Lobules were classified as previously described (Russo et?al., 1992) into types 1�C3 (L1�CL3) on cross-sections of normal breast stained with ALDH1A1 and hematoxylin. The number of ductules per lobule was counted on scanned sections, and the lobules were classified as L1 if they contained ��20 ductules, L2 if 21�C60 ductules, and L3 if >60 ductules. The percentages of ALDH1A1+ cells in each lobule cross-section was determined by counting the number of nuclei with surrounding positive staining and dividing by the total selleck chemicals llc number of nuclei. Mammosphere Culture Mammosphere culture was performed as previously described (Dontu et?al., 2003), at a density of 20,000 viable cells/ml in primary culture. Counting of mammospheres was done manually, after 7�C10?days, in six-well plates under light microscope in at least three wells for each condition. Statistical Analysis Data were analyzed using GraphPad Prism v6.0. Mann-Whitney U test or one-way ANOVA was performed to determine statistical significance, unless otherwise stated. P values?
