Immunofluorescence and Immunohistochemistry Protocols

Studies were performed as described previously [16] (link), [17] (link), [19] (link). The primary antibody for MELK (1∶200, Sigma-Aldrich, Missouri) was used to visualize the fluorescent signals using the following secondary antibodies: Alexa 488 or Alexa 555 (1∶1000, Cell Signaling Technology, MA). Specificity was determined using no-primary control slides. For immunohistochemistry, the following primary antibodies were used: Nestin (anti-Nestin, clone 10C2, 1∶200, mouse monoclonal antibody, MAB5326, MA) and Ki67 (anti-Human Ki-67, clone MIB-1, 1∶1, mouse monoclonal antibody, Dako, Denmark). The Envision system (Dako) followed by Diaminobenzidine (DAB) method was used for detection of primary antibody according the manufacturer’s protocol. For paraffin-embedded slides, hematoxylin was used as a nuclear counterstain. IHC scoring was performed using automated digital image analysis (ImageJ).