Ab129815

Formalin-fixed and paraffin-embedded tissue sections of 4 µm were dewaxed and subjected to antigen retrieval with the EnVision FLEX Target Retrieval reagent, high pH (DAKO, Hamburg, Germany). After the blocking step using peroxidase blocking solution (DAKO, Germany), immunohistochemical staining with anti-ALDH1 (clone 44, 611,194 BD Biosciences, dilution 1:000) or anti-ALDH1A3 (ab129815, Abcam, Cambridge UK, dilution 1:300) antibodies was done in an automated stainer (Dako Autostainer Plus, DAKO). Visualization of immunoreactivity was performed using the universal immuno-enzyme polymer method (Nichirei Biosciences Inc., Tokyo, Japan). Sections were developed in diaminobenzidine (Lab Vision Corporation, Fremont, CA, USA). As a control, a subset of slides was processed in parallel under the identical conditions except for the omission of anti-ALDH1 antibodies. Immunostaining results were evaluated by an experienced neuropathologist (CJS).

Samples of MCF7, MDA-MB-231, and PC-3 cells were lysed using a RIPA lysis buffer (sc-24948A, Santa Cruz Biotechnology, Dallas, TX, USA). Equal quantities of isolated proteins were then mixed with 5× Laemmli loading dye. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (12%) and electrophoretically transferred at a constant current of 25 V for 30 min using a Trans-Blot TurboTM Transfer System (Bio-Rad, Hercules, CA, USA) onto a polyvinylidene difluoride (PVDF) membrane, Santa Cruz Biotechnology). After blocking with 3% (w/v), fat-free milk diluted in TBST (50 mM Tris-HCl, 150 mM NaCl, pH 7.4, 0.1% (v/v) Tween 20) for 30 min at 4 °C, the membrane was incubated overnight at 4 °C with one of the following primary antibodies: anti-ALDH1A3 (ab129815; Abcam, Cambridge, UK) or anti-GAPDH antibody as a loading control (ab9485, Abcam). The amount of each antibody used was as per the manufacturer’s instructions. The next day, the primary antibody was removed, and the washing and blocking processes were carried out for 15 and 30 min, respectively. The secondary antibody (goat anti-rabbit IgG H&L (HRP) (ab205718, Abcam)) was then added for 90 min. The secondary antibody was then removed and the membrane was washed for 15 min to prepare it for imaging using a ChemiDocTM Imaging System (Bio-Rad, Hercules, CA, USA).

Three‐micrometer thick of paraffin‐embedded tumor or lung tissue sections were rehydrated and processed by standard procedure for HE staining and immunohistochemistry (IHC). The following primary antibodies were used: mouse anti‐vimentin 1/200 (clone V9, Thermo Fisher Scientific, Inc., Cambridge, UK); mouse anti‐CD34 1/100 (clone QBEND‐10, ab8536, Abcam, Cambrige, UK); rabbit anti‐CD31 1/50 (ab28364, Abcam); rabbit anti‐fibronectin 1/150 (ab2413, Abcam); rabbit anti‐ALDH1A3 1/100 (ab129815); rabbit anti‐MAP7 1/100 (NBP1‐84853). The EnVision system of labeled polymer‐HRP anti‐mouse or anti‐rabbit IgG (DakoCytomation, Agilent Technologies, Santa Clara, USA) was used undiluted as a secondary antibody. IHC of patients' tumor samples was scored 0, 1, or 2 to define absent, low or high expression of ALDH1A3 and MAP7, respectively.

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.

Western blotting was performed on ALDH^hi and ALDH^lo sorted cells using rabbit anti-human ALDH1A3 antibody (Ab 129815, Abcam, Cambridge, UK).