Approval of use of human specimens was given by the Emory University Institutional Review Board. All of the clinical samples were collected with informed consent under Health Insurance Portability and Accountability Act approved protocols.
Paraffin-embedded lung cancer with matched lymph node metastasis tissue array (LC814) was obtained from US Biomax, Inc. (Rockville, MD, USA). IHC analysis of RSK2 expression and stathmin phosphorylation was performed using tissue array samples as previously described.46 (link) In brief, human tissue sections were incubated in 3% hydrogen peroxide after deparaffinization and rehydration. Antigen retrieval was achieved by microwaving the sections in 100 mM Tris (pH 10.0) and 10 mM sodium citrate (pH 6.0) for RSK2 and phospho-stathmin S16 staining, respectively. The slides were subsequently blocked with 2.5% horse serum and avidin–biotin complex system (Vector Laboratories, Burlingame, CA, USA). The primary antibodies, anti-RSK2 antibody and anti-phospho-stathmin S16 antibody, were applied at a dilution of 1:100. Detection was achieved with 3,3′-diaminobenzidine and counterstained with hematoxylin. IHC staining results were scored as 0 for no staining, 1+ for weak staining, 2+ for moderate staining and 3+ for strong staining.
Paraffin-embedded lung cancer with matched lymph node metastasis tissue array (LC814) was obtained from US Biomax, Inc. (Rockville, MD, USA). IHC analysis of RSK2 expression and stathmin phosphorylation was performed using tissue array samples as previously described.46 (link) In brief, human tissue sections were incubated in 3% hydrogen peroxide after deparaffinization and rehydration. Antigen retrieval was achieved by microwaving the sections in 100 mM Tris (pH 10.0) and 10 mM sodium citrate (pH 6.0) for RSK2 and phospho-stathmin S16 staining, respectively. The slides were subsequently blocked with 2.5% horse serum and avidin–biotin complex system (Vector Laboratories, Burlingame, CA, USA). The primary antibodies, anti-RSK2 antibody and anti-phospho-stathmin S16 antibody, were applied at a dilution of 1:100. Detection was achieved with 3,3′-diaminobenzidine and counterstained with hematoxylin. IHC staining results were scored as 0 for no staining, 1+ for weak staining, 2+ for moderate staining and 3+ for strong staining.