Alexa fluor 647 conjugated goat anti rat

Immunofluorescence was performed as described previously (Oegema et al., 2001 (link); Monen et al., 2005 (link)), with a fixation time of 40 min in methanol at −20°C. The following primary antibodies were used: Alexa Fluor 488–conjugated mouse anti–α-tubulin (1:500, DM1α; Sigma-Aldrich, St. Louis, MO), Alexa Fluor 596-conjugated goat anti-GFP (used for GFP::EMR-1, 1:250, ab6660; Abcam, Cambridge, MA), monoclonal mouse anti-GFP (used for GFP::ASPM-1, 1:200, A11120; Invitrogen, Waltham, MA), rabbit anti-KLP-18 and rat anti-KLP-18 (1:10,000 and 1:200, respectively, gifts of Olaf Bossinger, RWTH Aachen University; Segbert et al., 2003 (link)), rabbit anti–ASPM-1 (1:5000; gift of Arshad Desai, Ludwig Institute for Cancer Research; Wignall and Villeneuve, 2009 (link)), rat anti-LMN-1 (1:500; gift of Katherine Wilson, Johns Hopkins University; Gruenbaum et al., 2002 (link)), and rabbit anti–MESP-1 (1:3000). Anti–MESP-1 antibody was raised through genomic antibody technology (Strategic Diagnostics, Newark, DE) against amino acids 60–159 and then affinity purified. Secondary antibodies used were Alexa Fluor 555–conjugated goat anti-rabbit and Alexa Fluor 647–conjugated goat anti-rat (both at 1:500; Invitrogen).