At the end of the injection courses, the mice were bled for their plasma.
Label
Cy3
Label protocol
After washing with phosphate-buffered saline (PBS)/3% fetal bovine serum/0.1% Tween 20, reactive antibodies were detected using Cy3-conjugated goat-anti-human or goat-anti-mouse IgG/IgM secondary antibody (diluted 1:4,000 in PBS/3% fetal bovine serum; Jackson Immunoresearch). After incubating for 45 minutes with the secondary antibody, the slides were washed with washing solution (PBS/3 % fetal bovine serum/0.1% Tween 20).
Hybridization protocol
Synovial antigen arrays were produced using a robotic microarrayer to print peptides and proteins on ArrayIt SuperEpoxy microscope slides (TeleChem International, Sunnyvale, CA). On each slide, antigens are printed at 0.2 mg/ml in four replicates. Slides were then blocked overnight (phosphate-buffered saline (PBS), 3% fetal bovine serum, 0.1% Tween 20) and incubated for 1h at 4°C with individual mouse plasma diluted at 1:200 in PBS/3% fetal bovine serum. Slides were washed with PBS, water-rinsed, and spun dry.
Scan protocol
Slides were then scanned to determine pixel intensities for each antigen feature (Genepix 4000B; Molecular Devices, Sunnyvale, CA, USA). Genepix Pro 5.0 software (Axon Instruments) at 532 nm was used to detect IgG and IgM reactivity. Images were saved as tiff files.
Description
1:200 dilution Plasma
Data processing
GenePix Pro 5.0 software (Axon Instruments) was used to determine the net median pixel intensities for each antigen feature (each value is background-corrected by removing the background around each spotted protein). The median intensities for each protein were centered around 300 and log-scaled. A level of 10 digital fluorescence intensity units was set as a threshold for significant reactivity. The formula for values below 10 is log(10/300,2), and for all other values it is log(median pixel intensity/300,2). Data analysis was performed using Significance Analysis for Microarrays (SAM) software (http://www-stat-class. stanford.edu /SAM/servlet/ SAMServlet) to identify antigen features with statistically significant differences in reactivities between the experimental groups. Cluster software was then used to hierarchically group the samples and antigen features on the basis of a pairwise similarity function, and TreeView software was used to display the data as a heat map (http://rana.lbl.gov/EisenSoftware.htm).
The median intensities for each protein were centered around 300 and log-scaled. A level of 10 digital fluorescence intensity units was set as a threshold for significant reactivity. The formula for values below 10 is log(10/300,2), and for all other values it is log(median pixel intensity/300,2).
