Figure 1.

Antibody validation and cell line expression of ALDH1 through Western blot and immunofluorescent staining followed by AQUA analysis on a series of control cell lines. A: The Western blot shows positive protein expression for ALDH1 in the cell lines CaCO, A431, and HT29 with a band at 55 kDA. No ALDH1 expression was detected in MB453, BT474, MCF7, 1650, and Mell888. β Tubulin serves as a control. B: Representative images of the three cell lines, MB175, A431, and HT29, with positive ALDH1 expression. The cell lines are represented on a tissue microarray; immunohistochemical staining was performed and the images were captured on the Cy5 channel with the PM1000 microscopy platform. Subsequent AQUA analysis resulted in an AQUA Score, which was used to define positive ALDH1 staining for breast cancer tissue. C: Immunohistochemical staining and AQUA analysis of ALDH1 were performed in fourfold redundancy on a control TMA consisting of cell lines, breast cancer samples, liver, and normal breast tissue. AQUA scores for each cell line are shown in fourfold redundancy. Cell lines showing positive expression for ALDH1 with an AQUA score above the threshold of 10 are MB175, A431, and HT29, thus confirming the Western blot results for ALDH1 positivity in A431 and HT29 cell lysates. The threshold of 10 was determined as the lowest AQUA Score for MB175 on these experiments performed in fourfold redundancy (no Western blot was performed for MB175 lysate).