Sera were obtained from 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
Green/red fluorescence intensities were acquired on an LI-COR Odyssey Imager (Lincoln, NE) at scanning intensities of 7/7 in both channels (700 nm/800 nm), 0.8 - 1.0 mm offset, at a spatial resolution of 21 µm. Staining of the Flag and HA control peptides that frame the arrays gave rise to high and homogeneous spot intensities with a coefficient of variation of <2%.
Description
pooled BXD2 sera (3 mice)
Data processing
Quantification of spot intensities and peptide annotation were based on the 16-bit grey scale tiff file that exhibits a higher dynamic range than the 24-bit colorized tiff file. PepSlide® Analyzer software was used to analyze the data. This program breaks down the fluorescence intensities of each spot into raw, foreground, and background signals, and calculates the standard deviation of the foreground median intensities.
Submission date
Jan 26, 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
Data table header descriptions
ID_REF
VALUE
Software computed foreground median intensities, standard deviation, and % deviation.
