Cy3 conjugated goat anti mouse secondary antibody igg igm

Antigens were diluted to 0.2 mg/ml in PBS or water and robotically spotted onto SuperEpoxy 2 Microarray Substrate Slides (ArrayIt) as described (56 (link)). A total of 330 RA-associated autoantigens were used, of which 52 are citrullinated, 263 are native, and 9 are control. Arrays were circumscribed using a hydrophobic Aqua-Hold Pap Pen 2 (Fisher Scientific). Chip was blocked overnight with PBS containing 3% FCS and 0.05% Tween 20. Arrays were probed with 1:300 diluted mouse sera, washed and incubated 1:2,000 dilution of Cy3-conjugated goat anti-mouse IgG+IgM secondary antibody (Jackson ImmunoResearch). Arrays were scanned using the GenePix 4000 scanner at wavelength 532 nm, and the median pixel intensities of the features and background values were determined using GenePix Pro version 3.0 software (Molecular Devices). Results were expressed as median fluorescence units, representing the median values from 4–8 identical replicates of an antigen on each array following subtraction of the median values of both intra-slide negative control (BSA) and inter-slide negative control (blank well features). Investigator that performed the array experiments was blinded to the experimental conditions used foreach sample.