Alexa 488 or 594 conjugated antibodies

Whole embryos were immersed in a solution of 4% paraformaldehyde in PBS for 1 hour at 4°C on a rocking platform. Embryos were frozen and cut into sections 10 μm thick. Pups and adults were anesthetized and perfused transcardially with 4% PFA in 0.12 m phosphate buffer, pH 7.4. After perfusion, eyes were removed from the skull and postfixed overnight in fresh fixative. Serial frozen sections were processed for immunocytochemistry using the antibodies listed in S1 Table Lectin PNA conjugates (1/50; L-21409, Molecular Probes, OR 97402) were also used. Alexa 488- or 594-conjugated antibodies (1:200, Invitrogen) were used for secondary detection. Nuclear staining was achieved in Hoechst 33342. Fluorescent images were obtained using an Olympus confocal microscope (FV-1200-IX83).

Mouse anti-TbSUMO (1C9H8) mAb (1∶2000), rabbit anti-TbRPA1 affinity-purified antiserum (1∶600) [4] (link) and rabbit anti-GFP polyclonal (1∶5000; Invitrogen) were used as primary antibodies. Goat anti-mouse and anti-rabbit Alexa 488 or 594 conjugated antibodies (Invitrogen) were used as secondary antibodies. Detailed subcellular localization and colocalization analysis was performed by deconvolution 3D microscopy as described previously [35] (link) (see Supporting Information Text S1).