Experimental design for screening anti-HEp-2 cell antibodies and identifying target autoantigens in SSc patients. HEp-2 cell proteins were extracted and separated on two-dimensional (2-D) gels. Total and enriched nuclear protein extracts were used as substrates for 2-D electrophoresis. One gel was stained with silver nitrate and used as the reference gel, and proteins of the 11 other gels were transferred onto polyvinylidene difluoride (PVDF) membranes. Membranes were immunoblotted at 1:100 dilution with pooled sera from 12 healthy blood donors or from sets of three patients with the same phenotype of systemic sclerosis (SSc). After immunoglobulin G (IgG) immunoreactivities were revealed, the 2-D immunoblots were stained with colloidal gold to visualize the transferred proteins. 2-D immunoblots were scanned before and after colloidal gold staining with the use of a densitometer, then analysed by using image analysis software, and finally compared with the reference gel. Selected protein spots were extracted from another gel stained with Coomassie brilliant blue, and candidate proteins were identified by mass spectrometry. Database searching was used to identify the antigens.