Detecting Anti-Nuclear Antibodies in Serum Samples

Individual serum from each treatment group was evaluated for the presence of anti-nuclear antibodies using HEp-2 ANA kit (Inova Diagnostics, Inc., San Diego, CA, USA) as previously described [28 (link)–31 (link)]. Manufacturer’s specific directions were followed in all procedures. Each serum sample was diluted 1:80 and incubated on HEp-2-fixed substrate slides for 1 h at room temperature in a humidified chamber. After three 5-min washes with PBS, the substrate slides were treated with a 1:100 dilution of Alexa 488 goat anti-mouse IgG (H + L) (Life Technologies) for 45 min at room temperature. After three washes, Vectashield DAPI mounting medium (Vector Laboratories, Burlingame, CA, USA) was applied, and overlaid with a glass coverslip. Fluorescence was detected by fluorescence microscopy at ×400 magnification by using a Nikon microscope, and all images were obtained with exposure of 200 ms.