Sera were obtained from B6 and BXD2 mice by retro-orbital eye bleeding. After separation from blood, sera were stored at -80°C.
Label
DyLight800
Label protocol
The secondary antibodies used for all arrays were from Rockland Immunochemicals, Gilbertsville, PA. They were conjugated to the DyLight dyes (DyLight680 or DyLight800) by the company.
Hybridization protocol
Peptide arrays were first blocked 60 min in Blocking Buffer for Near Infra Red Fluorescent Western blotting (Rockland Immunochemicals, Inc., Gilbertsville, PA). The microarray was washed twice in PBS, pH 7.4 with 0.05% Tween 20 (PBS-T), and incubated for an additional 30 min in washing buffer. The array was then incubated overnight at 4°C with mouse sera diluted 1:200 for anti-mouse IgG-specific analysis or 1:1000 for anti-mouse IgM-specific analysis (secondary Abs from Rockland Immunochemicals, Inc., Gilbertsville, PA). After multiple washes in washing buffer, the microarrays were incubated for 30 min with the secondary antibody in PBS-T at room temperature. After two additional washes in washing buffer, the microarrays were rinsed with ultrapure water and dried in a stream of air.
Scan protocol
LI-COR Odyssey Imaging System; scanning offset 1 mm, resolution 21 µm, scanning intensities of 5, 7 and 9 (main assays) and 5/7 (red/green, control assay)
Data processing
A software algorithm breaks down fluorescence intensities of each spot into raw, foreground and background signal, and calculates the standard deviation of foreground median intensities.
Submission date
Jan 23, 2015
Last update date
Feb 28, 2015
Contact name
Jennie Hamilton
Organization name
University of Alabama Birmingham
Department
Department of Medicine, Division of Clinical Immunology and Rheumatology
General Approach for Tetramer Based Identification of Autoantigen Reactive B Cells: Characterization of La and snRNP Reactive B Cells in Autoimmune BXD2 Mice
