Immunocytochemistry of Primary Neurons

For immunocytochemistry, primary neurons were fixed in pre-chilled methanol at −20 °C for 10 min, washed in PBS (3 × 5 min), permeabilized in 0.2% Triton X-100 (St. Louis, MO, USA) in 1× PBS (20 min at RT), and blocked for 1 h in 10% Normal Goat Serum (Roche Diagnostics) in 1× PBS. Primary antibodies were incubated overnight at 4 °C in blocking solution. The primary antibodies used were CERKL(ex2) and CERKL(ex5), and were obtained in-house against epitopes encoded in exon 2 or exon 5 of the mouse Cerkl gene respectively [16 (link)]. After incubation, coverslips with cells were rinsed in 1× PBS (3 × 5 min), incubated with the corresponding secondary antibodies conjugated to Alexa Fluor 488 (Life Technologies, Grand Island, NY, USA) (1:500) at RT (1 h) in blocking solution. Nuclei were stained with DAPI (Roche Diagnostics, Indianapolis, IN, USA) (1:1000), washed again in 1× PBS (3 × 5 min), and mounted in Mowiol 4–88 (Merck, Darmstadt, Germany).