Af488 conjugated streptavidin

Cryostat sections (7 μm) made from OCT (TissueTek) embedded dLNs were fixed with 2% PFA for 20 min, and then washed and blocked in blocking buffer (PBS with 1% BSA, 0.3% Triton-100, Fc-blocker and 5% rat serum and mouse serum). Sections were then stained in blocking buffer with biotin-labelled anti-mouse IgD (clone 11-26c, eBioscience) and PE-labelled anti-mouse BCL6 antibodies (clone K112-91, BD Biosciences), and subsequently stained with AF488-conjugated streptavidin (Thermo Fisher Scientific). Fluorescent images were captured using a 4× objective on a fluorescence microscope (Keyence).
For imaging the distribution of TLR7-NP in the dLNs, AF647-labelled TLR7-NP was s.c. injected into mice (n = 2) at the tail base. dLNs were harvested 48 h later. Tissue sections were stained with anti-mouse IgD_Al488 (clone 11-26c, SouthernBiotech), CD4_BV421 (clone GK1.5, BioLegend) and CD35_biotin (clone 8C12, BD Biosciences). Streptavidin_A555 (Invitrogen, catalogue number S32355, 1:100) was used to detect biotin. Images were captured using a 20× objective on a fluorescence microscope (Leica, DMi8).

Spleen tissue from mice treated with Alexa Fluor 647-labelled IL-2-Fc was collected 12 h post injection and frozen in optimal cutting temperature (OCT) embedding medium (Sakura). Tissue sections (5 μm) were cut using a Leica CM3050 S Cryostat, followed by fixation in acetone (7 min) and re-hydration in PBS. After blocking with Protein Block solution (Dako), sections were stained with anti-B220-biotin, PE-conjugated anti-FoxP3 and BV421-conjugated anti-CD4 (in Antibody Diluent solution, Dako), followed by washing in PBS+1% Tween 20 and staining with AF488-conjugated streptavidin (Thermo Fisher). Sections were mounted with Fluoromount (Sigma) and analysed using a Leica DM5500 microscope.

To characterize binding of the Zif-FLAP candidates, K562 or K562/CD25 cells were first adhered to the glass bottom of a tissue culture-treated cell imaging dish (Eppendorf, Hamburg, Germany) by gravity sedimentation^{42 (link)}. Briefly, the cells were washed twice with TBS and resuspended to 1 × 10⁶ cells/ml in TBS. Then, 400 μL of the cell suspension was pipetted onto the dish and incubated for 30 min at room temperature. The medium was aspirated and the cells were fixed in 4% paraformaldehyde at room temperature for 10 min and then treated with blocking solution (1% BSA in TBS) at room temperature for 1.5 h. Zif-FLAP candidates, which were biotinylated along with their chemical synthesis by a commercial facility (Biologica, Nagoya, Japan), were incubated in a zinc ion-containing buffer (0.5 mM DTT and 100 μM ZnCl₂ in TBS) at room temperature for approximately 5 min to ensure proper folding before dilution to a final concentration of 1 μM with TBS. The cells were then incubated with the Zif-FLAP candidates at 4 °C for 16 h. Binding was detected by first incubating the cells with 5 μg/mL AF488-conjugated streptavidin (Invitrogen) at 4 °C for 30 min. After replacement with TBS buffer, the cells were visualized under a laser scanning confocal microscope (Zeiss LSM 780, Zeiss, Jena, Germany) using a 488-nm argon laser.