Citifluor mounting media

Immunofluorescence was performed as described previously (Bayless et al., 2012 (link)). Cells were washed in PHEM buffer (60 mM 1,4-piperazinediethanesulfonic acid, 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 10 mM EGTA, and 2 mM MgCl₂, pH 6.9) and then fixed in formaldehyde fixative (3.2% paraformaldehyde and 0.2% Triton X-100, in PHEM buffer) for 5 min. Cells were washed three times in 0.1% BSA in PBS (BSA-PBS) before a 24-h incubation at 4°C with primary antibody diluted in 1.0% BSA-PBS. Primary antibodies used in this study were α-TtCen1 (Stemm-Wolf et al., 2005 (link)) and α-glutamylation (GT335; Adipogen; Wolff et al., 1992 (link)). Cells were then washed three times in 0.1% BSA-PBS before incubation for 2 h at 25°C in secondary antibody diluted in 1.0% BSA-PBS. Secondary antibodies used in this study were Alexa Fluor 488, 594, or 647 goat α–rabbit IgG, Alexa Fluor 488, 594, or 647 goat α–mouse IgG (Invitrogen). Cells were then washed three times in 0.1% BSA-PBS, and 1 µl of cell pellet was added to coverslip and mounted to a slide with Citifluor mounting media (Ted Pella). Samples were then sealed using clear nail polish.