Serial sections (5-μm-thick) were deparaffinized in xylene, treated with 0.3% hydrogen peroxide for 20 min, followed by incubation in protein-free block (Dako, Carpinteria, CA, USA) for 10 min to inhibit the nonspecific binding of the primary antibody.
Results Saturation and competitive binding studies employing the natural ligand uPA revealed specific binding of the primary mAb (Kd = 1.0 ± 0.1 nM). Assay specific IC50 values of the peptide-based, literature-known structures DOTA-AE105 and WX-360 were in the low micromolar range while no binding was detected for DOTA-AE105M (non-binding mutant) [3,4].
The binding of the primary antibodies was highly specific as demonstrated by the negative results of the controls (Figure 7B).
Alexa Fluor-conjugated secondary antibodies (Molecular Probes, USA) were used at a dilution of 1∶400 to immuno-localize the binding of the primary antibody.
In CD4/Foxp3 double immunostaining experiments, binding of the primary Ab (anti CD4) in the first label was detected using avidin-biotin alkaline phosphatase (Thermo Scientific, Rockford, IL, USA) and Ferangi Blu (Biocare, Concord, CA, USA) as chromogen.
Other residues that bind the primary amine of the glutamate ligand in mGlu1 (Thr188, Asp208, Tyr236 and Asp318) are less conserved in VKRs, but their chemical features are compatible with the binding of the primary groups of amino-acids.
The negative control staining in the absence of the primary antibody demonstrated that the baseline level was substantially dominated by non-specific binding of the primary antibody rather than that of secondary antibody or autofluorescence (data not shown).
The cell body staining was absent (not shown), which lead us to suspect that the hair bundle signal may have resulted from non-specific binding of the primary antibody.
Clover effectively identified motifs whose presence in the training sequences could not be explained by chance alone, regardless of whether these motifs actually facilitate binding of the primary factor.
were used to detect the binding of the primary antibodies.
Four different secondary antibodies were used to image binding of the primary antibodies.
