Immunohistochemical Analysis of Apoptosis Regulators

Paraffin-embedded tissue sections were hydrated using a descending alcohol series, incubated for 10–20 min in target retrieval solution (DAKO, Glostrup, Denmark) and incubated with blocking solution (2% normal goat serum/PBS). After permeabilization, sections were incubated overnight at 4 °C with mouse-anti Mcl-1 (1/200; Santa Cruz, Dallas, TX, USA), mouse-anti Bcl-x (1/50, BD Bioscience, Heidelberg, Germany), or mouse-anti- Hypoxyprobe™ Mab-1 antibody for detection of pimonidazole (Hydroxyprobe, Burlington, MA, USA). Antigens were detected with a horseradish peroxidase-conjugated secondary antibody (1/250) and 3,3′-diaminobenzidine staining (DAKO) as previously described [48 , 49 (link)]. Nuclei were counterstained using hematoxylin.