Mouse anti aldh1

The expression of selected breast cancer related proteins was analyzed with multicolor immunofluorescence staining. Therefore, 4 μm thick sections from compact spheres that were priorly formalin-fixed and paraffin-embedded were used. After retrieval with high pH solution (Dako, Glostrup, Denmark) in the microwave at 360 W for 10 min and blocking with normal goat serum for 30 min, samples were incubated with primary monoclonal antibodies such as rabbit anti-pan-cytokeratin, mouse anti-Ki67, mouse anti-Vimentin, mouse-anti human epithelial antigen (HEA), rabbit anti-Her2neu (all Dako) and mouse anti-ALDH1 (BD Bioscience) and mouse anti-CD44 (Thermo Scientific) for 1 hour at room temperature. After washing with PBS, slides were incubated for 1 hour with secondary fluorescent labeled antibody cocktail consisting of Alexa 488 goat anti-rabbit IgG and Alexa 594 goat anti-mouse IgG (life technologies, Carlsberg, CA, USA). Cells were washed again and slides were coverslipped with the SlowFade^® gold antifade mounting media with 4′,6-Diamidin-2-phenylindol (DAPI) (life technologies). Image analysis was performed with a fluorescent microscope Olympus Basic BX51 (Vienna, Austria).

Whole cell lysates were prepared using RIPA buffer (Cell Signaling Technologies, Danvers, MA) and protease inhibitor cocktail (Sigma-Aldrich). Cell lysates were quantified using the Bio-Rad DC Protein Assay (Bio-Rad, Hercules, CA). Eighteen μg of protein or 50 ng of indicated recombinant ALDH were loaded on 4–12% gradient NuPAGE Novex Bis-Tris gels (ThermoFisher, Carlsbad, CA) in MES SDS running buffer. Proteins were transferred to nitrocellulose membranes using the iBlot 2 Gel Transfer Device (ThermoFisher) and blocked in 5% blotting milk in TBST buffer (50 mM Tris pH 7.5, 150 mM NaCl, 0.05% Tween20). Membranes were incubated with either mouse anti-ALDH1 (BD Biosciences #611194, San Jose, CA), rabbit anti-ALDH1A2 (Abcam #ab156019, Cambridge, MA), rabbit anti-ALDH1A3 (Abcam #ab129815), rabbit anti-ALDH2 (Abcam #ab108306) or rabbit anti-ALDH3A1 (Abcam #ab129022) at 1:1,000 dilution and rabbit anti-β-Actin primary antibodies (Cell Signaling Technologies # 4970L) primary antibodies at 1:5,000 dilution overnight (4°C). HRP-conjugated secondary antibodies were used as follow: HRP-anti-rabbit IgG (Cell Signaling Technologies #7074S) or HRP-anti-mouse IgG (Cell Signaling Technologies #7076S) at 1:10,000 dilution, incubated (RT) for 1 hour, and visualized with SuperSignal West Dura Chemiluminescent Substrate (ThermoFisher) on a Bio-Rad Gel Doc XR+ Gel Documentation System.