Anti mouse f4 80 fitc antibody

Zebrafish embryos were euthanized with tricaine in E3 medium, fixed in fresh 4% paraformaldehyde in PBS and incubated in methanol (1 h at −20°C). Embryos were rehydrated using decreasing concentrations of methanol in PBST (PBS, 0.3% Triton X-100) and permeabilized using 1 μg/ml proteinase K at room temperature for 1 h. Larvae were then re-fixed and incubated with a cold ethanol/acetone solution (2:1) for 10 min at −20°C. Fixed and permeabilized embryos were then incubated in blocking solution (10 mg/ml BSA, 2% BFS, 1% DMSO, 0.1% Triton X-100) and later incubated either with the anti-mouse c-kit biotin-conjugated antibody (1:100, Abcam, #ab25022), the anti-mouse Ly-6G/Ly-6C (Gr-1) biotin-conjugated antibody (BioLegend, #108404), the anti-mouse Ki-67-FITC (1:100, Ebioscience, #11-5698-80) or the anti-mouse F4/80-FITC antibody (1:100, Ebioscience, #11-4801-82). Streptavidin-FITC (1:500, Ebioscience, #11-4317-87) was used as secondary antibody for biotin-conjugated primary antibodies. For quantification, the CHT of chimeric animals was visualized and manually counted for positive murine cells for a specific antibody on an epifluorescence microscope (Zeiss Axio Observer). Percentage was calculated with respect to the total murine CHT cell count in individual larvae (blue-labeled cells). Mean and s.e.m. was calculated from total larvae analyzed.

Bone marrow (BM) cells were isolated from the tibias and femurs of BALB/c mice, and the cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) low glucose (Gibco) supplemented with 10% FBS and 20 ng/ml GM-CSF. Cells were harvested on day 7 for further experiments. BM cells were identified by flow cytometry with an anti-mouse CD11b (APC) antibody (cat. no. 17-0112-82, eBioscience) and an anti-mouse F4/80 (FITC) antibody (cat. no. 11-4801-82, eBioscience). Cells were cultured in a humidified atmosphere of 5% CO₂ at 37 °C. In terms of cell transfection, BMDMs were inoculated at a density of 60~80% before the transfection experiment. After 24 h, cells were transiently transfected with the indicated vectors with Lipofectamine 2000 according to the manufacturer’s instructions. Then, the cells were harvested at different time points.