Slowfade gold antifade mountant

HeLa cells were plated on glass coverslips in 24-well plates and transfected with pCI/HA-GSK-3β; 48 h later, the cells were washed with PBS and fixed with 4% paraformaldehyde in PBS for 30 min at room temperature. After washing with PBS, the cells were blocked with 10% goat serum in PBS containing 0.2% Triton X-100 for 1 hr at 37 °C, and incubated with mouse anti-HA (1:400) and rabbit anti-β-catenin antibody (1:400) overnight at 4 °C. After incubation with secondary antibodies (Alexa 488-conjugated goat anti-rabbit IgG and Alexa 555-conjugated goat anti-mouse IgG, 1:1000, Invitrogen, Carlsbad, CA, USA) plus Hoechst 33342 at room temperature, the cells were washed with PBS, mounted with SlowFade® Gold Antifade Mountant (Invitrogen, Carlsbad, CA, USA), and visualized with a TCS-SP5 dual photon laser-scanning confocal microscope (Leica, Bensheim, Germany). We used the Image J software to quantify the fluorescence intensity of the nucleus and whole cell. The ratio of fluorescence level in the nucleus and the cytoplasm (total-nucleus) was calculated and presented as average of the cells from 4–5 fields at random.

For immunofluorescent staining of MHC-II, slides were dried O/N at 37°C tissue sections were de-paraffinated and rehydrated with xylene for 15 min, 100% ethanol for 30 s, 100% 1-propanol for 30 sec, 90% 1-propanol for 30 s, and 70% 1-propanol for 30 sec. To rehydrate, sections were soaked in 0.85% NaCl for 5 min, followed by TBS for 30 min. For antigen retrieval, slides were immersed in 37°C TBS for 20 min, then room temperature TBS for 20 min. Sections were blocked in 10% sheep serum/TBS for 30 min at room temperature in a humidified box. Sections were then incubated with 0.02 mg/ml mouse anti-ovine MHC Class II:FITC (Serotec, MCA2228F) in 10 mg/ml BSA/0.05% Triton X/TBS, and incubated O/N at 4°C. DAPI 1 was added for 30 min before sections were washed three times in 0.05% Tween20 TBS, then rinsed in TBS only. Sections were mounted with SlowFade Gold Antifade Mountant (Invitrogen S36936). The stained sections were viewed by fluorescent microscopy using an Olympus BX51 and then photographed using an Olympus digital camera. Cell counts were performed using Image J Fiji software by subtracting background, increasing contrast, creating a RGB stack, selecting either the epidermis, or the papillary dermis, determined by the smooth collagen and elastic fibers then measuring percentage area that stained positive in the green field.

To determine the optimal time for P2C-mediated delivery, adult females were injected with P2C-EGFP (Chaverra-Rodriguez et al. 2018 (link)) 24 hr before and 4, 24, and 48 hr after a bloodmeal. Adult female ovaries were dissected 72 hr post-blood meal, mounted on a slide in SlowFade Gold Antifade Mountant (Invitrogen) between two double layers of scotch tape to prevent the ovaries from being squashed by the plastic coverslip. The coverslip was sealed in place using nail polish on the edges of the slip and ovaries were visualized on Olympus BX41. All images were captured using 311ms exposure for 200X and 1030ms exposure for 40X images.