Samples, preincubated with 10% goat serum for 30 min to block unspecific binding, were incubated with primary antibodies (LRIG1, a polyclonal rabbit antibody, diluted 1 : 500 and for EGFR a polyclonal rabbit antibody, Santa Cruz Biotechnology, Santa Cruz, CA, USA diluted 1 : 100, overnight).
After the required washing steps, the membrane used to detect GRFT binding was incubated with anti-rabbit immunoglobulin conjugated with HRP and washed again, and both membranes were processed using an enhanced chemiluminescence Western blotting detection system (Thermo Scientific).
To examine the effects of amino acids on PLG binding to the pathogen surface, 5×107 Sterne spores in binding buffer were incubated with 2 µg of PLG and 50 mM amino acids for 1 h on a rocker platform.
To eliminate non-specific binding, supernatants were incubated with 50 µl protein A agarose and cleared by centrifugation.
To determine specificity of DNA binding, samples were incubated with or without 20 ng of unlabeled competitor DNA for 10 min at room temperature.
To block nonspecific binding, sections were incubated with normal goat serum diluted 1∶50 in 5% bovine serum albumin dissolved in Tris-buffered saline (BSA/TBS).
In order to avoid nonspecific binding, cells were incubated with 0.5 µg Fc blocker (per 106 cells) for 30 minutes on ice followed by staining with rabbit anti-mouse MMP-9.
After three washes with TBS-T to remove excessive primary antibody binding, blots were incubated with horseradish peroxidase (HRP conjugated secondary antibody (1∶5,000) for 1 hr at room temperature.
2×105 cells in 100 µl of binding buffer were incubated with 2 µl of annexin V-PE and 2 µl of 7-amino-actinomycin D for 15 min at 25 °C in the dark.
For protein binding, cells were incubated with human IgG-Fc fusion proteins or with MuHV-4 glycoprotein-specific mAbs (1h, 4°C), washed ×2 in PBS, and then incubated with fluorescein-conjugated rabbit anti-mouse IgG pAb (Dako Cytomation) or phycoerythrin-conjugated goat anti-human IgG-Fc pAb (Sigma Chemical Co).
To control for specific binding, tissues were incubated with TUNEL reaction mixture that lacked labelled nucleotides.
