Anti vegf a antibody

Staining was performed on 24-well culture plates. After removing culture media, cells were fixed with methanol (4°C for 10 min), rehydrated with PBS and blocked with a donkey serum (dilution 1:20 in PBS), followed by an overnight incubation with prolactin antibodies (dilution 1:30 in PBS/2% rat serum, Santa Cruz, CA, USA) at 4°C. The secondary antibody (TRITC, donkey anti-goat, Dianova) was diluted 1:100 in PBS (pH 8.2) containing a 2% rat serum and was incubated for 30 min. After three rinses with PBS, the cells were blocked with goat serum (dilution 1:20 in PBS, 10 min), followed by incubation with an anti-VEGF-A antibody (dilution 1:30 in PBS/2% rat serum, Santa Cruz) overnight at 4°C. The secondary antibody (FITC, goat anti rabbit, Linaris) was diluted 1:50 in PBS (pH 8.2+2% rat serum) for 30 min. The cells were incubated with KCl (200 mmol) for 5 min and then with DAPI (0.2 μg/ml PBS pH 7.0, Sigma) for 1 h and finally mounted with medium for fluorescence analysis (Vector Laboratories, Burlingame, CA, USA).
For negative controls phosphate-buffered saline (PBS, Dulbecco, w/o Ca⁺⁺, w/o Mg⁺⁺) containing 2% rat serum replaced the primary antibody. Rabbit IgG (Dako) and goat IgG (Dianova) were applied at equal concentrations as the primary antibody.

After deparaffinization and rehydration, the slides with tissue sections (4 μm) were washed twice with PBS and then blocked for 1 h at RT with 2% (w/v) BSA in PBS containing 0.1% (v/v) Triton X100. The slides were subsequently incubated at 4 °C overnight with anti-ITGA6 (1:100 dilution) (Abcam) and anti-ITGB4 (1:100) (Santa Cruz Biotechnology; Dallas, TX, USA) antibodies for double recognition or anti-VEGFA antibody (1:100) (Santa Cruz) for single recognition in a humidity chamber. Probe ligation was performed using DUOlink (OLINK Biosciences; Uppsala, Sweden), as recommended by the manufacturer. Images were taken using a LSM710 confocal laser scanning microscope and ZEN software (Carl Zeiss; Oberkochen, Germany). Fluorescence images were quantified by measuring red-colored area using Image J (version 1.50i).