Dylight 488 conjugated donkey anti goat igg

Samples were fixed in paraformaldehyde (4% w/v in 1× PBS) for 15 min. Samples intended for labeling of secreted proteins (namely ALB and OPN) were treated with brefeldin A (10 µg/ml, R&D Systems, 1231/5) for 2 hr prior to fixation. Fixed samples were permeabilized with Triton X-100 (0.25% v/v in 1× PBS) for 10 min and incubated in blocking buffer (5% v/v donkey serum and 0.1% v/v Triton X-100 in 1× PBS) for 1 hr at room temperature. We incubated samples for 1 hr at room temperature or overnight at 4°C with one or two of the primary antibodies listed in Table 2 diluted in blocking buffer. The next day, we incubated samples for 1 hr at room temperature with one or two of the following secondary antibodies diluted in blocking buffer: DyLight 488-conjugated donkey anti-rabbit IgG (1/50 from stock, Abcam, ab96919), DyLight 550-conjugated donkey anti-mouse IgG (1/50 from stock, Abcam, ab98767), and DyLight 488-conjugated donkey anti-goat IgG (1/50 from stock, Abcam, ab96935). Samples were mounted in Fluoromount G with DAPI (Southern Biotech, 0100–20) and imaged no earlier than the day after mounting using an Axiovert 200M microscope (Carl Zeiss, Inc.) and associated Zen Pro software. In order to capture entire arrays as one image for later analyses, we utilized the tiling feature of Zen Pro.