Goat anti mouse igg1 alexa 647

Antibodies and dilutions used were as follows: chicken CD3 (clone CT3, Southern Biotech, Birmingham, Alabama, USA), 1:100 for FACS; Bu-1 (clone AV20, Southern Biotech), 1:1,000 for FACS, 1:300 for immunofluorescence; CD45 (clone AV53), 1:1,000 for FACS and immunofluorescence; chicken CSF1R [20 (link)], 1:100 for immunofluorescence; GFP Rabbit IgG Antibody Fraction, Alexa Fluor 488 Conjugate (Invitrogen, Carlsbad, CA, USA), 1:500 for immunofluorescence; Goat-anti-mouse-IgG1-alexa 647 (Invitrogen), 1:5,000 for FACS; Alexa Fluor 546 F(ab')2 Fragment of Goat Anti-Mouse IgG (Invitrogen), 1:500 for immunofluorescence.

Flow cytometry was performed to characterise the CSF1R-transgene-expressing cells using mouse monoclonal antibodies to chicken CD3 (clone CT3, AbD Serotec), Bu-1 (clone AV20, AbD Serotec), CD45 (clone AV53, Institute for Animal Health, UK), KUL01 (SBA, SouthernBiotech), CSF1R (Garcia-Morales et al., 2013 (link)) and MHC II [clone 2G11 (Kaufman et al., 1990 (link))]. Cells were stained for 30 min at 4°C, washed with PBA (PBS, 0.5% BSA and 0.05% sodium azide) followed by incubation with a goat-anti-mouse-IgG1-alexa 647 (Invitrogen) for 30 min at 4°C. Cells were washed with PBA and analysed using a FACS Calibur flowcytometer (BD Biosciences). At least 100,000 events were acquired in the lymphocyte gate and data were analysed using the software program FlowJo (Threestar).