Anti survivin antibody

Cells were incubated for 24–48 h with and without treatments and then harvested for western blot analysis. Briefly, cells were incubated in lysis buffer for 20 min on ice and centrifuged at 17,000× g for 10 min at 4 °C. Protein concentration of cell lysates was determined with BCA reagent (Thermo Fisher Scientific, Houston, TX, USA). Lysates were separated on 8–16% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. Immunoblots were probed with anti-survivin antibody (Novus; 1:2000, Centennial CO, USA) in 5% BSA overnight at 4 °C and HRP labeled secondary antibody was added for 1 h at RT. The signal was developed on X-ray films. The uncropped western blotting figures can be found in Figure S2.

Immunohistochemistry was performed on paraformaldehyde-fixed-paraffin-embedded samples according to the manufacturer’s instructions (Novus biologicals, Littleton, USA) as described previously [22 , 23 (link)]. As primary antibody a polyclonal rabbit anti-Survivin antibody (dilution 1:1000, Novus Biologicals, Littleton, USA) was used. The Ki-67 staining was carried out using a monoclonal anti-Ki-67 cell cycle marker (BioLogo, Kronshagen, Germany) as primary antibody. Visualization of survivin and Ki-67 was performed with the Vectastain ABC Kit (Vector Burlingame, USA) and UltraVision Detection System Large Volume DAB Plus Substrate System (Thermo Scientific, Fremont, USA) according to the manufacturer’s instructions. Hematoxylin was used for counter-staining. Evaluation of immunohistochemistry was performed by light microscopy in a total magnification of 1:400. Survivin expression was determined by using a special Java-based image processing and analysis program (Image J, W. Rasband, National Institutes of Health, USA). Survivin expressing cells were colored brown and considered positive while cells exhibiting a blue staining were counted as negative. The percentage of brown area per crypt was calculated, averaged and statistically evaluated.