Alexa647 labeled ova

Alexa647-labeled OVA (Thermo Fisher Scientific, Middletown, VA) with or without SF-10 was administrated intranasally into BALB/c mice as described above. At 15 min after vaccination, the nasal tissues were isolated and cryosection slides were prepared [12 (link), 18 (link)]. The slices were then stained with Hoechst33342 (Doujin Chemical Co., Kumamoto, Japan) and fluorescence images were acquired with a microscope (BX-X700; Keyence Co., Osaka, Japan). At 60 min after vaccination, nasal cells were also isolated by Accutase^®, stained with anti-mouse CD326, CD11c and CD8α Ab (BioLegend), and analyzed by flow cytometry.

Alexa647-labeled OVA (Thermo Fisher Scientific) was formulated using different combinations of adjuvant components in Glu/PBS buffer. Mice were immunized i.d. at the tail base. After the indicated times, the fluorescent intensities of Alexa647 at the injection site and the draining inguinal lymph node were measured using a VISQUE InVivo Smart-LF in vivo imager (Vieworks, Seoul, South Korea). Mice were administered a low fluorescent diet (iVid-neo; Oriental Yeast, Tokyo, Japan) prior to at least 7 days before the experiment to reduce abdominal background fluorescence as much as possible. This was done because many plant-based ingredients used in regular mouse chows contain chlorophyll components (mainly alfalfa) that emit between 675 and 685 nm, which usually disturbs the signal detection in the abdominal area [28 (link)].