Alexa fluor 647 anti mouse igg

WT and mutant fish were fixed in 4% paraformaldehyde overnight at 4°C before removal of the eye, which was incubated in 30% sucrose in PBS overnight at 4°C. Eyes were then embedded in OCT compound (Tissue Tek O.C.T.; Sakura, 4583) and cryosectioned at 20 µm (Leica). Slides were rehydrated in PBS before blocking for 1 h at room temperature using 150 µl blocking solution (1% sheep serum, 5% bovine serum albumin, 0.3% Triton X-100 and 0.1% Tween-20 in PBS). Slides were then incubated with 150 µl primary antibody solution (4C4; a generous gift from Noemie Hamilton, Department of Biology, University of York, York, UK; 1:50 dilution in block solution; antibody registry ID: AB_10013752) overnight at 4°C. After incubation, slides were washed three times in PBS for 20 min followed by incubation with secondary antibody (Alexa Fluor 647 anti-mouse IgG; Invitrogen, A-21235) for 2 h. Slides were then washed in PBS three times for 20 min, before adding Fluoroshield with DAPI (Sigma-Aldrich, F6057-20ML) and applying a glass coverslip. Slides were imaged on a Zeiss LSM 900 confocal microscope using a 40× water-immersion objective.

For the GVGH study, GAS strain GAS51∆M1 was grown ON in Todd Hewitt broth plus Yeast extract at 37 °C and 5% CO₂. Bacteria were pelleted at 4000× g for 5 min and washed with PBS. Bacteria were then blocked with PBS + 3% (w/V) BSA for 20 min and incubated with pooled mice or rabbit sera diluted 1:10,000 in PBS  +  1% (w/V) BSA for one hour.
For the NIBSC study, GAS strain NCTC 8198 was grown in Todd Hewitt broth plus Yeast extract at 37°C and 5% CO₂ until reaching OD₆₀₀ 0.4. Bacteria were centrifuged at 4000× g for 5 min, and the resulting pellet was washed with PBS. Bacteria were then blocked with PBS + 10% (V/V) goat serum for 20 min and incubated with pooled mouse sera diluted 1:4 in PBS + 10% (V/V) Goat serum and 0.1% (w/V) BSA at 4 °C for one hour.
After washes, samples were incubated with Alexa Fluor 647 anti-mouse IgG (Molecular Probes), Alexa Fluor 488 anti-rabbit IgG (Molecular Probes) or FITC anti-mouse IgG (Thermofisher Scientific, Waltham, MA, USA) diluted in PBS + 0.1% (w/V) BSA for 45 min at 4°C. Finally, bacteria were fixed with 4% (w/V) formaldehyde for 20 min.
Flow cytometry analyses were performed on a FACS Canto II flow cytometer (BD Biosciences). Results are reported as overlaid histograms with the relative fluorescence intensity on the X axis and the percentage of the maximum number of events on the Y axis.

DNA replication was analyzed by examination of BrdU incorporation in one-cell embryos at 4, 6, 8, and 10 hpi. BrdU (Roche) was added to the KSOM to a final concentration of 10 μM. After incubation at 38°C for 30 min, the embryos were washed three times in 1% BSA/PBS and fixed with 3.7% PFA/PBS for 1 h at room temperature. After fixation, the samples were washed with 1% BSA/PBS three times, then washed three times in PBS containing 0.05% Tween-20. The samples were then placed under 2 N HCl containing 0.1% Triton X-100 for 1 h at 37°C. Next, the samples were washed with 1% BSA/PBS three times and transferred into 0.1 M Tris–HCl (pH 8.5)/PBS containing 0.02% Triton X-100 for 15 min at room temperature. The samples were washed three times with 1% BSA/PBS and incubated overnight with primary antibodies: mouse anti-BrdU (1:100; Roche) and rabbit anti-H3K9me3 (1:1,000; Upstate/Millipore). Alexa Fluor 647 anti-mouse IgG (Molecular Probes) and fluorescein isothiocyanate–conjugated donkey anti-rabbit (Jackson ImmunoResearch Inc.) were used as secondary antibodies; slides were prepared as described above.

HCT116 cells were seeded at 1 × 10⁵ cells/well in a six-well plate and grown for 2 days. In case of inducing the expression of OsTIR1(WT or F74G) under the control of the Tet promoter (Supplementary Fig. 1F), cells were treated with 0.5 µg/mL of doxycycline for 24 h, and then 100 µM IAA or 1 µM 5-Ph-IAA was added. For detecting EGFP and Clover signals after ligand treatment, cells were trypsinized and fixed in 4% methanol-free paraformaldehyde phosphate buffer (FUJIFILM Wako Pure Chemical Corporation) at 4 °C overnight. Fixed cells were washed and resuspended in PBS containing 1% BSA. To detect OsTIR1-V5 in Supplementary Fig. 1F, fixed cells were treated with anti-V5 antibody (Invitrogen, #R960-25) and subsequently stained with Alexa Fluor 647 anti-mouse IgG (ThermoFisher, #A-21236). For measuring the DNA signal after ligand treatment, cells were trypsinized and fixed in 70% EtOH. Fixed cells were washed, resuspended in PBS containing 1% BSA, 50 μg/ml of RNase A, and 40 μg/ml of propidium iodide, and incubated at 37 °C for 30 min. Flow cytometric analysis was performed on a BD Accuri C6 machine (BD Biosciences) using FCS4 Express Cytometry software (DeNovo Software). Ten thousand cells were analyzed from each sample, except in the case of Supplementary Fig. 1F, in which 50,000 cells were analyzed.