Alexa fluor 488 conjugated anti rabbit igg antibody

Immunofluorescence was performed as described previously 38 (link). In brief, cells were transfected with miR-204-5p mimics or inhibitors and then fixed with 4% para-formaldehyde, permeabilized with 0.5% Triton X-100, blocked with 1% BSA in PBS, and subsequently incubated with primary antibodies. The primary antibodies were as listed in Table S4. The cells were then incubated with Alexa Fluor 488-conjugated anti-rabbit IgG antibody (#4412, Cell Signaling Technology, Danvers, MA, USA) and Alexa Fluor 594-conjugated anti-rabbit IgG antibody (#8889, Cell Signaling Technology) at room temperature for one hours in the dark. The cells then washed with PBS and counterstained with DAPI (Invitrogen) for 5 min following the manufacturer's protocol. Images were obtained with a laser scanning microscope (Carl Zeiss, Jena, Germany).

The isolated CD4⁺ T cells were seeded in a 24-well plate at the density of 1.5 × 10⁶ cells/well and treated as described in the “Flow cytometry” section. The In situ expressions of RORγt, IL-17A and IL-17F in the treated CD4⁺ T cells were examined via immunofluorescence staining. The treated cells were fixed in cold methanol at 4°C for 10 min. Then, the cells were washed with PBS and blocked with 5% BSA in PBS for 30 min. The blocked cells were incubated with rabbit anti-RORγt (Santa Cruz biotechnology, Santa Cruz, CA, USA), rat anti-IL-17A (Cell Signaling, Danvers, MA, USA) and goat anti-IL-17F (R&D Systems, Minneapolis, MN) antibodies for 3 h at 4°C. Subsequently, the cells were incubated with a donkey anti-goat IgG-PE antibody (Santa Cruz biotechnology, Santa Cruz, CA, USA), a Alexa Fluor 488-conjugated anti-rat IgG antibody, a Alexa Fluor 488-conjugated anti-rabbit IgG antibody or a Alexa Fluor 647-conjugated anti-rabbit IgG antibody (Cell Signaling Technology, Inc.) for 1 h at 37°C. In addition, 6-diamidino-2-phenylindole (DAPI) or propidium iodide (PI) staining was performed for 5 min at the same temperature. The cells were detected using the Leica DMRA2 fluorescence microscope with FW4000 software (Leica, Germany). Positive cells or negative cells under 10 fields of view selected randomly for each group were counted for average percentages.