Biotinylated anti mouse igg

Cell lysates (1 × 10⁵ cells) were separated on a polyacrylamide gel using the Laemmli discontinuous buffer system. Proteins (ERK, p-stat5, and AKT, 1 × 10⁵ cells/ lane; p-ERK, STAT5, p-AKT, MLC2, and p-MLC2, 4 × 10⁵ cells/ lane) were loaded onto a 5–20% or 10–20% SDS-PAGE gel and transferred onto a PVDF membrane. The membranes were blocked with 5% BSA (Sigma-Aldrich) in Tris-buffered saline (TBS) with 0.1% Tween-20 (TTBS) and incubated overnight at 4°C with the following Abs in 5% BSA-TTBS: phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) rabbit mAb (1:1000), p44/42 MAPK (Erk1/2) rabbit mAb (1:1000), phospho-Stat5 (Tyr694) rabbit mAb (1:1000), Stat5a mouse mAb (1:1000), phospho-Akt (Ser473) rabbit mAb (1:1000), Akt rabbit mAb (1:1000), MLC2 rabbit mAb (1:1000), and phospho-MLC2 (Thr18/Ser19) rabbit mAb (1:1000). All Abs were obtained from Cell Signaling Technology. After thorough washing, the membranes were incubated with biotinylated anti-rabbit IgG (1:2500) (Zymed Laboratories) or biotinylated anti-mouse IgG (1:2500) (Zymed) for 1 h at RT and reacted with extra-avidin peroxidase (Sigma-Aldrich) (1:2000) for 15 min at RT. The avidin–peroxidase conjugate was detected on the basis of enhanced chemiluminescence (Luminata Forte Western HRP Substrate; Millipore) using the Lumino image analyzer LAS-4000 (Fuji Film).

Tumor tissues were fixed in 10% formalin and embedded in paraffin. Immunohistochemical staining was carried out using anti-HER-2/neu (Dako, Glostrup, Denmark) as the primary antibody for c-erbB-2. After making slices using a microtome, tissue sections (4 μm) were immersed in xylene solution to remove residual paraffin and hydrated in an alcohol series. Sections were boiled for 5 minutes to retrieve antigenicity in citrate buffer (pH 6.0) and left for 30 minutes at room temperature. After exhausting endogenous peroxidase for 10 minutes with H₂O₂ in methyl alcohol, sections were washed thrice with phosphate-buffered saline (PBS). Sections were blocked for 30 minutes with blocking solution (Histostain kit, Zymed Company, San Francisco, CA, USA) at room temperature. Sections were then incubated with anti-HER-2/neu (1 : 200, Dako) at room temperature. After rinsing thrice with PBS, sections were incubated with biotinylated anti-mouse IgG (1 : 300; Zymed). After washing, sections were incubated with avidin-alkaline phosphatase for 7 minutes at 40°C. Sections were visualized with red chromogen at 40°C and counterstained using the Mayer hematoxylin method. Sections were mounted and observed under light microscopy.