Ki67 gtx16667

Primary antibodies were obtained from the following sources: DUSP6 (ab76310) from Abcam (Toronto, Canada), β‐actin (MAB1501R) from Millipore (Oakville, Canada), ERK2 (sc‐154) and MEK2 (sc‐524) from Santa Cruz Biotechnology (Santa Cruz, CA), phosphorylated ERK1/2 (M8159) from Sigma‐Aldrich (Oakville, Canada), phosphorylated MEK1/2 (9121) from Cell Signaling Technology (Danvers, MA, USA), chromogranin A (20085) from ImmunoStar (Hudson, WI), proliferating cell nuclear antigen (PCNA; 18197) from Abcam and Ki67 (GTX16667) from Genetex (Irvine, CA). Horseradish peroxidase antibodies were obtained from GE Healthcare Life Sciences (Mississauga, Canada) and alkaline phosphatase‐conjugated antibodies from Promega (Madison, WI). For immunofluorescence, Alexa Fluor 488 conjugated antibodies were obtained from Molecular Probes (Waltham, MA). Other materials were purchased from Sigma‐Aldrich unless stated otherwise.

Protein expression in PDX tumor tissues was detected using IHC. Paraffin-embedded PDX tumor tissues were cut into 8-μm sections and preincubated in 3% H₂O₂ and 0.3% Triton X-100 before microwaving for antigen retrieval. For detection of Ki-67 protein immunostaining, sections were microwaved in Tris buffer (pH 6) for 30 min. The antigenicity of the tumor cells in sections was blocked in 5% horse serum (Chemicon, Temecula, CA, USA) for 30 min and incubated with a diluted (1:200) Ki-67 (GTX16667, GeneTex)-specific antibody for 2 h at room temperature. Staining was developed using the streptavidin-biotin-peroxidase method and an LSAB 2 kit purchased from Dako (Carpinteria, CA, USA). Briefly, sections were washed in phosphate-buffered saline (PBS) and incubated with a biotinylated anti-rabbit secondary antibody. The samples were rewashed in the same buffer and incubated with a streptavidin-biotin-peroxidase complex. Staining was completed after incubation with substrate-chromogen solution. The incubation duration in solution with 3,3′-diaminobenzidine was determined using low-power microscopic inspection. Slides were washed, dehydrated, and coverslipped using a mixture of di-styrene, plasticizer, and xylene (DPX mounting medium) (44581, Sigma-Aldrich). Adjacent sections on the same slides were counterstained with hematoxylin for general histological orientation.