TMA slides were deparaffinized and rehydrated according to common protocols. Antigen retrieval was performed by boiling the slides in citrate buffer (pH = 6.0) for 10 min using a microwave oven. Sections were blocked using solution of 10% FBS (Thermo Fisher Scientific) and 1% BSA in TBS for 2 h at room temperature and probed with pERK1/2 antibodies (Cell Signaling Technology, D13.14.4E, 1:100 in TBS with 1% BSA) overnight at 4 °C. Slides were rinsed with 0.025% Triton-X100 (Fisher Scientific) in TBS, probed with secondary HRP-conjugated anti-rabbit IgG (Cell Signaling Technology, 7074, 1:1000 in TBS with 1% BSA) for 1 h at room temperature and developed with a 3,3-diaminobenzidine kit (Vector Laboratories, Burlingame, CA, USA). Separate slides were counterstained with hematoxylin (Fisher Scientific). After staining slides were rinsed with DI water, dehydrated and mounted using toluene (Fisher Scientific).
Tissue slides with tumor samples obtained from mice injected with PEO4 cells (see below) were stained at the histology core at the University of Michigan using EDTA-based antigen retrieval and mouse anti-ALDH antibody (BD Biosciences, San Jose, CA, USA, clone 44/ALDH; 1:100) as previously described [43 (link)]. For stain quantification, five sections from three tumors per treatment group were analyzed by two people. Counts were then compared using a 2-sided Student’s t test.
Tissue slides with tumor samples obtained from mice injected with PEO4 cells (see below) were stained at the histology core at the University of Michigan using EDTA-based antigen retrieval and mouse anti-ALDH antibody (BD Biosciences, San Jose, CA, USA, clone 44/ALDH; 1:100) as previously described [43 (link)]. For stain quantification, five sections from three tumors per treatment group were analyzed by two people. Counts were then compared using a 2-sided Student’s t test.
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