Ventana benchmark automated staining system

HMEC-1 cells and lung endothelial tissue from experimental rats were fixed using 10% formaldehyde for 2 h at 37°C and then embedded in paraffin. For HMEC-1 cells, rat anti-human SEMA-7A antibody (1:1,000, cat no. ab23578; Abcam) labeled with fluorescein isothiocyanate (green fluorescence) for 12 h at 4°C was used to analyze SEMA-7A expression prior to and following treatment with Anti-SEMA-7A (2 mg/ml), SEMA-7A (2 mg/ml) or PBS (2 mg/ml) for 2 h at 4°C. Lung tissues were cut into sections 4-µm thick. Antigen retrieval was performed in tissue sections and the sections were incubated with the following primary antibodies: SEMA-7A (1: 1,000; cat no. ab23578), M1-7 (1:1,000; cat no. ab61108) and FSF/FKF (1:1,000; cat no. ab200478; all Abcam) for 8 h at 4°C and correlative secondary antibodies: HRP-conjugated goat anti-rabbit IgG mAb (1:2,000; cat no. PV-6001; ZSGB-BIO, Beijing, China) were applied for specimens for 24 h at 4°C. The staining of the slides was performed with the avidin-biotin-peroxidase complex. A Ventana Benchmark automated staining system (Version 3.0, Ventana Medical Systems, Inc; Roche Diagnostics, Basel, Switzerland) was used to analyze SEMA-7A, ERF, ERK, M1-7 and FSF/FKF levels using confocal microscopy (Carl Zeiss LSM780, Carl Zeiss AG, Oberkochen, Germany).

Tumors from NSCLC carcinoma xenograph mice treated with AbMTA2 or PBS were excised on day 30 and fixed using 10% formaldehyde followed by embedding in paraffin wax, and cut into serial sections (4 µm). Tissues were washed with PBS-Tween-20 (PBST) three times at room temperature and antigen retrieval was performed on the tumor sections using a microwave heating AR (43 (link)) subsequent to a series of ethanols (100, 95 and 80%). Tumor sections were washed with PBST at room temperature and incubated with primary antibodies: EGF (1:500; cat. no. ab9695), and VEGF (1:500; cat. no. ab32152) (both from Abcam) at 37°C for 2 h. Then, sections were blocked with 5% BSA (Sigma-Aldrich; Merck KGaA) at 37°C for 2 h and horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody (Bio-Rad Laboratories, Inc.) was used to incubate primary antibodies at a 1:5,000 dilution at 37°C for 2 h. A Ventana Benchmark Automated Staining System (Ventana Medical Systems, Inc., Tucson, AZ, USA) was used for the observation of protein expression.