Ab16901

Rat hippocampal neuronal primary cultures were prepared from embryonic day 19 (E19) rat hippocampi as previously described [50 (link)]. Primary hippocampal cultures were transfected with GFP at DIV9-10 and fixed at DIV15 with 4% PFA/sucrose in PBS for 10 min at room temperature (RT), then washed three times with PBS. Neurons were permeabilized with 0.1% Triton X-100 in PBS for 15 min at RT. After incubation with the blocking solution of the PLA kit (Duolink® PLA Technology), cells were incubated o/n at 4 °C with primary antibodies against CAP1 (1:100, Abnova, H00010487-M02) and cofilin1 (1:100, Cell Signaling, 5175). According to the manufacturer’s instructions, after two washes with Wash Buffer A, secondary antibodies conjugated with oligonucleotides were added and the oligonucleotides of the bound probes were ligated and amplified by a fluorescent polymerase that visualizes the PLA clusters. Coverslips were then washed three times with decreasing concentration of Buffer Solution B. After this, cells were labeled with primary antibody against GFP (1:500, Millipore, AB16901) for 1 h at RT, washed, and then incubated with secondary antibody. After three washes with PBS, coverslips were mounted on slides in Fluoromount™ Aqueous Mounting Medium (Sigma-Aldrich).