Cy3 anti rat antibody

Indirect in situ immunofluorescence microscopy to detect Tub1 was performed as previously described (Kilmartin and Adams, 1984 (link)) using a rat anti-α-tubulin (YOL1/34, Oxford Biotechnology, 1:100 dilution). An anti-rat Cy3 antibody (Jackson Laboratory) was used as a secondary antibody. Cells were scored on a Zeiss Axio Observer Z1 inverted microscope (Zeiss. Thornwood, NY).≥100 cells were counted per time point.

For MaSCs doublet analyses, mammospheres were mechanically dissociated and single cells were allowed to divide once for 30 h in stem cell medium additioned with 20% methylcellulose in order to avoid aggregation. Cells were then transferred to a poly-lysine coated glass slide (Corning) and fixed with 4% paraformaldehyde (PFA) for 20 minutes at room temperature. Then, cells were blocked in donkey serum diluted 1:5 (20%) for 15 min, and incubated with anti-CD49f antibody (working dilution 1:250; clone GoH3, cat. no 555734, BD Pharmigen) for 1 h at room temperature. For Supplementary Fig. 6d, cells were further stained with a monoclonal Numb antibody as described in Colaluca et al.^{51 (link)}. Cells were washed with PBS, and later incubated with anti-rat cy3 antibody (working dilution 1:400; Jackson ImmunoResearch) for 30 min at room temperature. Following that, cells were washed again with PBS, and fixed with 4% PFA for 10 min at room temperature. After permeabilization with 0,1% Triton-X, cells were counterstained with DAPI (Sigma). Confocal microscopy was performed on a Leica TCS SP2 confocal microscope. A 63 × oil-immersion objective lens (HCX Plan-Apochromat 63× NA 1.4 Lbd Bl; Leica) was used for analysis.