Anti pegfr

TNBC cell xenograft tumor tissues were fixed in 10% neutral-buffered formalin and embedded in paraffin. The sections (5 μm thick) were deparaffinized in xylene for 2 min 3 times, rehydrated in graded alcohols for 1 min, and washed in distilled water. IHC staining was performed using the Lab Vision automated system (Thermo Fisher) through the MD Anderson Cancer Center Division of Surgery Histology Core. The slides were then incubated with anti-Ki-67 (#RM-9106, 1:100, Thermo Fisher), anti-pHER2 (#2243, 1:320, Cell Signaling), anti-HER2 (#APA342AA, Biocare Medical, Pacheco, CA, USA), anti-EGFR (#4267, 1:50, Cell Signaling), anti-pEGFR (#API300AA, Biocare Medical), anti-Bim (#2933, 1:200, Cell Signaling), and anti-cleaved poly(ADP-ribose) polymerase (PARP, #5625, 1:100, Cell Signaling). Immunostained slides were scanned using an Aperio AT2 slide scanner (Leica, Buffalo Grove, IL, USA) and captured at 20× magnification using ImageScope software (Leica Biosystems, Wetzlar, Germany). Then intensity was quantified by ImageJ software [19 (link)].

Paraffin sections of tumor samples were deparaffinized in xylene and rehydrated in a series of graded alcohols. Antigens were retrieved in 0.1 M EDTA-Tris buffer. Samples were incubated in 0.9% H2O2 for 5 min, followed by a one-hour blocking period in 1% BSA in PBS. Slides were incubated for one hour at room temperature with anti-Ki67 (1:100; Biocare Medical: Concord, CA, USA) and anti-pEGFR (1:100; phosphorylated Tyr1068 epidermal growth factor receptor; Biocare Medical: Concord, CA, USA), washed, and incubated in MACH1 Universal HRP-polymer (Biocare Medical: Concord, CA, USA) for one hour at room temperature. Then, samples were developed with Betazoid DAB Chromogen Kit (Biocare Medical: Concord, CA, USA), counter-stained with hematoxylin, and mounted with synthetic resin solution. Sections were evaluated using an optical microscope (magnification of 400× and 1000×) (Zeiss, Mod. Axioscope: Oberkochen, Germany). Images were acquired with a digital camera (5MP high-speed color; AmScope Mod. Mu500: Irvine, CA, USA).