Alexa fluor antibody labeling kit

A custom antibody production service (Bioyear, China) was utilized to generate rabbit polyclonal antibodies. The epitopes sequences of zebrafish Ddx4, Piwil1, Sycp3, and Pcna are listed in Table 1. All antibodies were purified from anti-serum using antigen-affinity chromatography as described previously (Figure 1A) (43 (link)). Briefly, 10 mg purified antigen protein was coupled to 2 mL cyanogen bromide (CNBr) activated agarose (Sangon Biotech, China). Forty mL of antiserum was purified by 2mL of antigen-coupled activated agarose. For Western blot analysis and single antibody immunofluorescence, the polyclonal antibodies were diluted in 50% glycerol in PBS and stored at -80 °C. For fluorescent labeling, the antibodies were stored in PBS at -80 °C. For immunofluorescence co-staining, the antibodies of Sycp3, Pcna and Ddx4 were conjugated with Alexa Fluor 488, 568 and 680, respectively using Alexa Fluor^® Antibody Labeling Kits according to the manufacturer’s manual (Thermo Fisher).

For surface staining, single cell suspensions were stained with anti-CD3 (UCHT1), anti-CD4 (RPA-T4), anti-CD161 (HP-3G10) antibodies (all from eBioscience, Germany). To analyze Foxp3 and CREM expression, cells were fixed and permeabilized with a FOXP3 staining buffer set (eBioscience, Germany) following the manufacturer’s instructions and stained with anti-Foxp3 (PCH101) antibodies (eBioscience, Germany), monoclonal anti-CREM (Abcam, Great Britain) or IgG isotype control antibodies for 30 min. Monoclonal anti-CREM antibodies and IgG isotype control antibodies were labeled with Alexa Fluor Antibody Labeling Kits (Thermo Fisher Scientific, USA) according to manufactures instructions. For measurement of intracellular cytokines, cells were treated with propidium iodide (P/I) and GolgiPlug (BD Bisciences, Germany) for 5 h and fixed and permeabilized with FOXP3 staining buffer set (eBioscience, Germany) following the manufacturers’ instructions. Intracellular cytokines were stained with anti-IFN-γ (4S.B3) APC and anti-IL-17 PE (64DEC17) (both eBioscience, Germany) antibodies.

Monoclonal antibodies were first labeled with Fluorochrome using Alexa Fluor™ Antibody Labeling Kits (ThermoFisher Scientific, Waltham, MA, cat # A20181 or A20186) and then 1 µg was used to label 10⁶ cells in 100 µl 1X PBS, 0.1% glucose and 2% FBS (cell wash buffer) for 30 min at 4 ^oC. Following labeling, cells were washed twice with cell wash buffer and resuspended in 100 µl cell wash buffer. Flow cytometric analyses were performed in a Quanteon Flow Cytometer (Agilent, Santa Clara, CA). Flow gating strategies were illustrated in Supplementary Fig. 2. Data was analyzed with FlowJo Software v10.8. Stable transfected cells stained with FM5148-Alexa Fluor 488 were sorted on a MA900 cell sorter (Sony Biotechnology, San Jose, CA). Sorting strategies were demonstrated in Supplementary Fig. 3a and b. Cells with high expression of HLA-DQα (FM5148-Alexa Fluor 488) were sorted and plated in RPMI-1640 + 10% FBS with the corresponding selection antibiotics.

Experimentally treated primary Th17 or memory CD4⁺ T cells were harvested by centrifugation at 1500 rpm for 3 min in polystyrene 5-ml round-bottomed flow cytometry tubes. Thereafter, cells were washed with 1X PBS prior to fixation in 4% formaldehyde for 15 min at room temperature. For surface-staining with fluorophore-conjugated CD45-RO, CCR7, CD25 and CD69 antibodies, fixed cells were first washed with 1X PBS then incubated with these antibodies for 45 min in the dark and at room temperature. For intracellular staining, fixed cells were permeabilized by a sequential treatment with 0.2% Triton-X-100 for 10 min and incubation with 1X BD Perm/Wash buffer (BD Biosciences) for 15 min at room temperature. Following a 15-min blocking step with a non-specific IgG, permeabilized cells were immunostained for 45 min in the dark and at room temperature with fluorophore-conjugated antibodies against cyclin D3, Ki67, total CDK9, cyclin T1, pThr186 CDK9, pSer175 CDK9, pSer2 CTD RNA polymerase II, or pSer529 p65 NF-κB. For antibodies that were unconjugated, Alexa Fluor antibody labeling kits from ThermoFisher Scientific were used for fluorophore conjugation. After immunostaining cells were rinsed with 1X PBS and then subjected to flow cytometry analysis using the BD LSR Fortessa instrument (BD Biosciences) equipped with the appropriate laser and filters.