Anti mouse ki 67 fitc

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.

Cells cultured in vitro or isolated from tumors or spleens were processed for surface labeling with appropriate antibodies. Propidium iodide was used to distinguish the live cells. Cells were further permeabilized using 70% ethanol or True-Nuclear™ transcription factor buffer set (cat# 424401, BioLegend) and stained for Ki-67 or FOXP3. Data were acquired using the Beckman CytoFLEX flow cytometer and analyzed using FlowJo software. Antibodies used for flow cytometry were purchased from Biolegend (anti-mouse CD16/32, cat#101320; CD8a Alexa Fluor^® 647, cat#100724; NK 1.1 Alexa Fluor^® 647, cat#108720; Ly-6G/Ly-6C (Gr-1) Alexa Fluor^® 488, cat#108417; CD19 FITC, cat#152404; CD45R/B220 Alexa Fluor^® 647, cat#103226; F4/80 Alexa Fluor^® 488, cat#123120; CD206 Alexa Fluor^® 647, cat#141712; FOXP3 Alexa Fluor^® 488, cat#126406; CD4 Alexa Fluor^® 647, cat#100530; CD11c Alexa Fluor^® 488, cat#117311; H-2K^b/H-2D^b FITC, cat#114606; I-A/I-E Alexa Fluor^® 647, cat#107618; CD80 Alexa Fluor^® 647, cat#104718; CD86 FITC, cat#105006; CSF-1R Alexa Fluor^® 488, cat#135511) and eBioscience (anti-mouse Ki-67 FITC, cat#11-5698-82; CD3ε FITC, cat#11-0031-82; CD11b eFluor 660, cat#50-0112-82).