Hep 2 human epithelial cell substrate slides

The procedures were performed as previously described [24 (link)–28 (link)]. Mouse sera were serial diluted by 2-fold from 1:40 to 1:1280 in PBS and mounted onto the HEp-2 human epithelial cell substrate slides (INOVA Diagnostics), which were then incubated at room temperature for 1 hour. After washing with PBS, the slides stained with 1:100-diluted Alexa Fluor568–conjugated goat anti-mouse IgG (Invitrogen) for 1 hour at room temperature, and washed with PBS. The stained slides were imaged using a Zeiss wide-field fluorescence microscope (Zeiss) at 400× magnification and analyzed using the corresponding Zeiss software. The fluorescence staining intensity was measured using the ImageJ 1.50i software, and the titer of the autoantibody was determined based on the negative staining control with PBS.

HEp-2 human epithelial cell substrate slides (INOVA Diagnostics) were incubated with 1:40 diluted mouse sera, washed, and incubated with fluorescence-conjugated anti-mouse IgG. The stained slides were observed and imaged at 400× magnification using an inverted wide-field fluorescence microscope (Zeiss). Quantification of the fluorescence intensity was carried out using the ImageJ 1.50i software. Briefly, Images with single stained color were converted into grayscale (8-bit) and the grey color was segmented properly using thresholding. Integrated density was analyzed to determine the fluorescence intensity of the staining.

Serum ANA was assessed using HEp-2 human epithelial cell substrate slides (INOVA Diagnostics), according to the manufacturer’s instructions. After staining, the images were acquired on an inverted wide-field fluorescence microscope (Zeiss) at 400× magnification and processed using Zeiss software (ZEN blue edition).

Sera collected from the mice were diluted 1:40 and subjected to ANA measurements using HEp-2 human epithelial cell substrate slides (INOVA Diagnostics) following the manufacturer’s instructions. The slides were observed and imaged under an inverted wide-field fluorescence microscope (Zeiss) at 400× magnification. The images were further processed with the Zeiss software (ZEN blue edition), and the fluorescence intensity of the staining was quantified with the ImageJ 1.50i software as described in 2.6.