Goat anti mouse igg2b 647

For immunohistochemistry, organoids were fixed for 1 hour in 4% paraformaldehyde, incubated in 30% sucrose at +4°C for 24h, embedded in 30% sucrose/OCT mixture (1:1) and frozen on dry ice.
Immunofluorescence staining was performed on 10-12 m cryostat sections as described previously (Theil, 2005) Invitrogen; 15240-062). The cells were grown for an average of 4-6 days until cultures reached around 80% confluence, before they were fixed for 15 min at room temperature in 4% paraformaldehyde/DPBS. Cultures were blocked for 45 min with blocking buffer (BB) containing 6% Goat serum (Dako, S-100) in DPBS, and subsequently incubated with Mouse-anti-Tra-1-60 (1/100, Santa Cruz, sc-21705) antibody diluted in BB for one hour. Cultures were permeabilised with 0.1% Triton-X in DPBS for 10 min, followed by an overnight incubation at 4˚C with Rabbit-anti-NANOG (1/800, Cell Signalling, #3580S) and Mouse-anti-OCT3/4 (1/250, Santa Cruz, sc-5279) antibodies diluted in BB supplemented with 0.1% Triton-X. Primary antibodies were detected with Goat-anti-Rabbit-488 (1/1000, Invitrogen, A11008), Goat-anti -Mouse-IgG2b-647 (1/1000, Invitrogen, A21242), Goat-anti -Mouse-IgM-555 (1/1000, Invitrogen, A21426) secondary antibodies.
Fluorescent images were captured using a Zeiss observer Z1 microscope.