Ab33483

CD45⁺CD3⁺CD4⁺CD25⁺Foxp3⁺ Tregs, CD45⁺CD11b⁺CD64⁺ macrophages, and CD45⁺CD11b⁺CD64^-CD11c⁺MHC II⁺ dendritic cells were sorted from the lung tissues. Cells were firstly centrifuged in a Cytospin centrifuge (800 rpm, 5 min, Thermo Shandon Cytospin 3) onto Cytospin slides which were air-dried. Fixed cell-slides were then coated in 4% paraformaldehyde (PFA) for no more than 10 min and carefully washed 5 min with sterile PBS three times. After fixation, coated-cells were subjected to a permeabilization with 0.1% Triton X-100 in PBS for no more than 10 min, washed with sterile PBS for 5 min, then embedded with blocking solution for 1 h at room temperature. The plants were then covered with Foxp3 (13-5773-82, eBioscience), CD11c (ab33483, Abcam), CD68 (ab213363, Abcam), p35 (ab131039, Abcam), and EBI3 (sc-166158, Santa Cruz Biotechnology), followed by embedding with secondary antibody (A21208, A32727 and A21094, Invitrogen) for 1 h and incorporated with 4',6-diamidino-2-phenylindole (DAPI) for 5 min.

The IMQ-induced psoriasis-like mice were divided into two groups with same manipulations as described in Section 2.4.6. Subsequently, mice were sacrificed at 1, 4, 8, 24 and 48 h post administration. Mice dorsal skins were freshly excised into small pieces and washed in PBS with three times, each time lasted for 5 min. Immunostaining was performed to find out the colocalization of DCs with NPs. Vertical sections were cut at the thickness of 10 μm. Then skin sections were fixed with 4% paraformaldehyde for 15 min, following by 5% BSA incubation for 1 h at ambient temperature, and then incubated overnight at 4 °C with anti-CD11c primary antibody (ab33483, Abcam, 1:200). After 1 h incubation with goat anti-mouse IgG H&L (ab150115, Abcam, 1:500) at room temperature, nuclear staining was performed using Hochest 33342 (10 μg/mL). Skin sections were eventually observed by using CLSM at an excitation wavelength of 488 nm and emission wavelengths between 500 and 530 nm for DiO channel detection. Immunofluorescence was observed at an excited wavelength with an Alexa Fluor® 647 filter.