Prolong gold antifade containing dapi

Primary human macrophages were grown on glass coverslips, infected with M. bovis BCG (BCG-lux) and treated as described, rinsed with PBS, fixed with methanol and permeabilized with 0.1% Triton X-100 (Sigma)56 (link) before being stained with Nile red 1:10,000 (dissolved in isopropanol), rinsed with water and then mounted with ProLong Gold antifade containing DAPI (Invitrogen). Images were acquired on a Zeiss LSM880 confocal microscope (Plan-Apochromat × 63/1.40 Oil-immersion lens) and analysed with Zen 2010 (Carl Zeiss), and fluorescence per cell measured by Volocity software.

For the cells cultured to differentiate, the fusion index was determined as reported earlier [25 (link)]. Briefly, coverslips were stained with the primary antibodies desmin and myogenin (see Table 2 for details) followed by the secondary antibodies goat anti-rabbit 568 (catalogue #A11036) and goat anti-mouse 488 (catalogue # A11029), and mounted with Prolong-Gold-Antifade containing DAPI (catalogue #P36931, Invitrogen), as described [25 (link)]. Fusion index was calculated as the percentage of desmin-positive nuclei within myotubes (containing three or more nuclei) divided by the total number of desmin-positive nuclei.

Internalization of fluorescently labeled dendriplexes was investigated by fluorescence microscopy. Cells were incubated with BDEF33 or AE2G3 complexes with or without amiR-155-FAM and alone as stated above. Then, cells were washed twice with 2 mM EDTA solution in PBS and fixed with a mixture of ethanol:glacial acetic acid (3:1, v/v). Cells were resuspended thoroughly to avoid clumps after fixation step and placed on slides.
Then, the slides were analyzed using phase-contrast microscopy for cell cytoplasm and nuclei visualization in transmitted light. After that, slides were mounted with Pro Long Gold antifade containing DAPI (Invitrogen MP, Waltham, MA, USA) to prevent dye photo-bleaching and identify cell nuclei further.
Phase-contrast, as well as fluorescent microscopy, was performed with the Axioscope 40 fluorescence microscope (Zeiss, Germany) equipped with a high-pressure mercury lamp HBO 50W, with Zeiss interference filter sets (Set No. 49 for DAPI) and CCD-chamber AxioCam 503 mono (at 1936 × 1460 px resolution and 14-bit capacity). DAPI-stained nuclei images and FAM signals were captured separately with the software package ZEN-2012 (Zeiss, Germany) on the magnitude X1000 (Figure S1). Exposure time was adjusted automatically.

22Rv1 cells were grown on 12 mm glass coverslips placed into a 24-well plate. After attachment, cells were starved overnight. The PKH67 labeled (green) exosomes (20 μg/mL) from TBX2 modulated PCa cells were incubated for 8 h with starved cells. Thereafter, cells were washed with PBS (3 times) and fixed with 4% paraformaldehyde for 10 min at RT, followed by 3 washes with PBS. The glass coverslips containing the fixed cells were mounted on glass slide using ProLong gold-antifade containing DAPI (Invitrogen, Eugene, OR, USA). Z-stack TD images were acquired using Nikon A1 R confocal microscope at the Imaging Core Facility of Texas Tech University Health Sciences Center, Lubbock, TX, USA.

Skin and muscle from the site of vaccination were isolated and fixed in 4% paraformaldehyde and 10% sucrose in PBS at 4 °C for 30 min to overnight. Tissues were further embedded in Tissue-Tec^® O.C.T (Sakura Finetek, Torrance, CA, USA) above liquid nitrogen. Cryosections of 4 μm were cut from the frozen tissue blocks and mounted on poly-l-lysine-coated slides. Slides were dried and stored at −20 °C until use.
Slides were brought to RT and placed in PBS for 5 min to remove the OCT and stained with rabbit anti-GFP (Abcam, Cambridge, UK). Anti-rabbit FITC (Abcam) was used as a secondary antibody. Slides were mounted with Prolong Gold antifade containing DAPI (Invitrogen, Carlsbad, CA, USA). Staining was evaluated by fluorescence microscopy.