Confocal imaging system

To analyze DC localization after an intravenous injection, BALB/c mice-derived imDCs labeled with CellVue Claret were injected into C57BL/6 mice via the tail vein. At 1, 7, or 14 days after adoptive transfer, the mesenteric lymph nodes and spleens were harvested. After being stained with DAPI (Beyotime, Shanghai, China; 1:1,000), tissue sections were observed under a confocal imaging system (Perkin Elmer, Waltham, MA, USA).
To investigate the survival of donor-derived DCs in recipient spleen and their expression of HO-1, untreated, CoPP-treated, or SnPP-treated BALB/c imDCs (5 × 10⁶) labeled with Cellvue Claret were adoptively transferred into C57BL/6 mice via the caudal vein. 7 or 14 days later, the spleens were harvested and then processed as frozen sections. The sections were incubated overnight with rabbit anti-HO-1 primary polyclonal Ab at 4°C and followed by a secondary antibody (FITC-conjugated goat anti-rabbit secondary mAb) for 30 min at 37°C. The nuclei were stained with DAPI (Beyotime, Shanghai, China; 1:1,000). The expression of HO-1 in Cellvue Claret-labeled DCs was evaluated by a confocal imaging system (Perkin Elmer, Waltham, MA, USA), viewing a minimum of five random microscopic fields.

Dendritic cells were washed twice with PBS and fixed with 4% polyoxymethylene for 30 min, then permeabilized with 0.1% Triton X-100 in PBS for 30 min at room temperature and blocked for 1 h with 2% BSA at 37°C. To detect HO-1, cells were incubated overnight with rabbit anti-HO-1 primary polyclonal Ab and APC-conjugated anti-mouse CD11c mAb at 4°C. The cells were then washed with PBS, and a secondary antibody (FITC-conjugated goat anti-rabbit secondary mAb) was applied for 30 min at 37°C. The nuclei were stained with DAPI (Beyotime, Shanghai, China; 1:1,000). Color images were captured under a confocal imaging system (Perkin Elmer, Waltham, MA, USA).

The target cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized by 0.25% TritonX-100 for 10 minutes and sequentially incubated with indicated primary antibodies, secondary antibodies (DyLight 649 goat anti-mouse IgG antibody, DyLight 488 donkey anti-rabbit IgG antibody) (Biolegend, USA), and 4-6-diamidino-2- phenylindole (DAPI) (Invitrogen, USA). Images were detected by the confocal imaging system (PerkinElmer, USA). The fluorescence intensity was analyzed by ImageJ software.