Detection of anti-nuclear antibodies in the sera of MRL/MpJ mice injected with plasmid DNA. Sera from the MRL/MpJ group injected with pcDNA3E were further analysed for anti-nuclear antibodies (ANA) using an indirect immunofluoresence assay routinely used for clinical diagnosis. Acetone-fixed human larynx carcinoma (HEp-2) cells (Bion Inc., IL, USA) were used to detect ANA, and the staining pattern in cell nuclei was identified using a modified version of a method, which has been previously described.26 Serum samples were diluted 1 in 40 in PBS with sodium azide (0.01% w/v). 20 μl of diluted MRL/MpJ sera were added to HEp-2 slides, and incubated for 30 min at room temperature in a humidified chamber. Slides were rinsed once in PBST followed by two 10-min washes. 10 μl of FITC-conjugated rabbit anti-mouse immunoglobulins (Dako A/S, Glostrup, Denmark), diluted 1 in 40 in PBST, were added and slides incubated for 30 min in a humidified chamber. Slides were mounted (in 70% glycerol, 2.5% DABCO in PBS, pH 8.3) and analysed using an epifluorescence microscope (magnification ×400) with filters optimized for FITC detection. (a) Positive control anti-nuclear antibody immunoreactivity with serum from a 15-week-old MRL/MpJ-Faslpr mouse. (b) A typical negative pattern of serum immunoreactivity found in saline-injected MRL/MpJ mice. (c and d) Two typical patterns of ANA immunoreactivity found in MRL/MpJ mice injected with plasmid showing fine-speckled granular and anti-nucleolar patterns of immunoreactivity, respectively.