Mouse Autoantibody Detection by ELISA

Quantification of serum Ig isotypes was performed by ELISA as previously described^{40 (link)}. Tissue sections from gender matched Rag1^−/− mice were used to detect mouse autoantibodies. Briefly, organs from the Rag1^−/− mice were dissected, fixed with neutral buffered formalin, embedded with paraffin and sectioned. After deparaffinization with EZPrep buffer (Ventana Medical Systems) and antigen retrieval with Cell Conditioning Solution (CC1) (Ventana Medical Systems) the sections were blocked for 30 minutes with Background Buster solution (Innovex), followed by Avidin/Biotin blocking for 8 minutes, mouse serum (1:50 dilution) incubation for 5 hours and biotinylated horse anti-mouse IgG (Vector Labs) incubation for 1 hour. The detection was performed with Streptavidin-HRP (Ventana Medical Systems) followed by incubation with Tyramide Alexa Fluor 488 (Invitrogen). The slides were then counterstained with DAPI (Sigma Aldrich) for 10 minutes, mounted, scanned with a Mirax scanner and visualized with Pannoramic Viewer (3DHISTECH). Scanned images were scored and representative snapshots were processed with Photoshop (Adobe) to switch the green and red channels for presentation purpose.