Immunofluorescence Staining of rHcABHD

Freshly collected female and male adults (20 ~ 30 for each) were washed, dehydrated, fixed, embedded and cut into cryostat sections as previous described (22 (link)). To block non-specific binding, the cryosections were treated with 10% normal goat serum in PBS containing 0.1% Tween-20 (PBST) at room temperature for 1 h. The sections were then incubated with rat anti-rHcABHD IgG (1:200) or normal rat IgG (control) at 4°C overnight. After three washes in PBST, the sections were then incubated with Cy3-labeled goat anti-rat IgG (1:500) for 1 h at 37°C. Following three washes in PBST, 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI, Sigma-Aldrich) were used for DNA staining at room temperature for 5 min. The samples were then immersed into anti-fade medium (Sigma-Aldrich) to prevent fluorescence fading for microscopic examination. Finally, the sections were imaged using LSM710 fluorescence microscope (Zeiss, Jena, Germany) at 60× magnification and digital images were analyzed using ZEN 2012 software (Zeiss).