Mib 1 ki 67 antibody

IHC staining for ER, PR, HER2, and ASAP1 was performed on 4 μm thick TMA sections using the Benchmark XT automated staining system (Ventana Medical Systems, Tucson, AZ, USA). The primary antibodies for ER, PR, and HER2 were as follows: ER (SP1) rabbit monoclonal primary antibody (Roche, Basel, Switzerland), PR (1E2) rabbit monoclonal primary antibody (Roche), HER-2/neu (4B5) rabbit monoclonal primary antibody (Roche), and anti-ASAP1/DDEF1 antibody (7B12) (ab125729, Abcam, Cambridge, UK; diluted 1:200). Heat-induced epitope retrieval was performed with CC1 Tris-EDTA buffer (Ventana Medical Systems, Tucson, AZ, USA) at 100 °C for 80 min. Detection of primary antibody was performed with the OptiView DAB IHC Detection Kit (Ventana Medical Systems). IHC staining for Ki-67 was performed on 4 μm thick TMA sections using the Bond-Max automated immunostainer (Leica BioSystems, Newcastle, UK). Monoclonal (MIB-1) Ki-67 antibody (M7240, Dako, Santa Clara, CA, USA; diluted 1:100) was also used for IHC. Heat-induced antigen retrieval was performed with Bond epitope retrieval solution (Leica BioSystems) after deparaffinization. Detection of primary antigen was performed using the Bond Polymer Refine Detection kit (Leica BioSystems) and 3,3’-diaminobenzidine tetrahydrochloride as a chromogen.

Materials for tissue cultures were purchased from Euroclone (Milan, Italy). Antibodies against p-Akt Ser473 [sc-135651], p-Akt Thr308 [sc135650], p-Ser2448 mTOR [sc-293133], p-Ser 37/46-4E-BP1 [sc-293124], and pSer235/23-S6 [sc-293144] were purchased from Santa Cruz (Santa Cruz, CA, USA). MIB1 (Ki67) antibody was purchased from Dako (Carpenteria, CA, USA). Autophagy assay kit (ab139484) was purchased from Abcam (Cambridge, UK). ApopTag^® peroxidase in situ apoptosis detection kit was purchased from Merck Millipore (Milan, Italy). RES529 was provided by Diffusion Pharmaceuticals, Inc. Bevacizumab and sunitinib (Suni) were obtained from the pharmacy of the San Salvatore hospital in L’Aquila. RES529, bevacizumab, and sunitinib were used as described [16 (link),18 (link),42 (link)]. Necrostatin-1 [Methylthiohydantoin-DL-tryptophan, CAS 4311-88-0, 51] and the ferrostatin-1 were purchased from Sigma Aldrich (St Louis, CA, USA).