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).
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).
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