Wga alexa flour 633

Fluorescent staining and imaging were carried out as previously described (Davidson et al., 2004 (link)). The following fluorescent dyes were used: 4-amino-5-methylamino-2′,7′-difluorescein diacetate (DAF-FM DA) from Thermo Fisher Scientific (Waltham, MA, United States) was used to detect presence of NO; wheat-germ agglutinin WGA Alexa Flour 633 (Thermo Fisher) was used to stain mucus; and, Cell Tracker Orange (Thermo Fisher) was used to label host tissues. The confocal fluorescent microscopy was performed using a Zeiss LSM 710 confocal microscopy (Carl Zeiss AG, Jena, Germany), stationed at the University of Hawai’i, Mānoa (UHM), Kewalo Marine Laboratory.

Mammary samples for histological analysis were prepared as previously described [8 (link), 14 (link)]. Briefly, paraffin sections were stained with haematoxylin and eosin (H&E) and cryosections were stained with phalloidin-TRITC (Sigma) to visualize filamentous actin (F-actin) and 4′,6′-diamidino-2-phenylindole (DAPI) (Invitrogen, Carlsbad, CA, USA) to visualize nuclear DNA.
Gall bladder and urinary bladder whole mounts were pinned onto a silicone pinning pad (SYLGARD 184 Dow Corning), fixed with 2.5% formaldehyde and stained with phalloidin-TRITC and DAPI or WGA Alexa Flour 633 (ThermoFisher W21404) and Sytox Orange (Invitrogen) as previously described [21 (link)].
Tissue sections were imaged with a Nikon Eclipse E400 epifluorescence microscope with an Olypus DP70 camera and images were merged using Adobe Photoshop software. Confocal images were acquired using a Leica TCS SP2 laser scanning spectrum confocal system (Leica Microsystems) and images were merged using Leica confocal software.