Ap conjugated anti mouse secondary antibody

CONCEPT telencephalon-eye organoids were fixed in 4% PFA for 15–30 min at room temperature and processed for immunostaining. The detailed information on primary antibodies is found in the Key Resources Table. Primary antibodies were visualized using Alexa Fluor 488-, 568-, and 647-conjugated secondary antibodies and imaged using a Zeiss AxioObserver Z1 microscope. When the sample did not fit in one image, multiple images were stitched to obtain an overview. In dual-color immunocytochemistry for PAX2 and FGF8, FGF8 was visualized using AP-conjugated anti mouse secondary antibody (Invitrogen A16038), and PAX2 was detected by biotin-conjugated anti rabbit secondary antibody (Invitrogen B2770) followed by HRP-conjugated streptavidin (Thermo Fisher Scientific 21130). For dual color immunocytochemistry of CNTN2 and FGF8 (both are mouse antibodies; no working antibodies raised in different species are available), immunocytochemistry of CNTN2 was performed first and visualized using HRP-conjugated streptavidin. Then, the samples were fixed in 4% PFA for 15 min to avoid antibody crosstalk. After that, FGF8 antibody was applied and then detected by an AP-conjugated secondary antibody. Brown and blue colors are well separated in Figure 5K, indicating that antibody crosstalk did not exist.