Alexa 647 conjugated anti rabbit igg

Neuroblastoma cells (N2a) grown on coverslips were fixed using 4% paraformaldehyde and 4% sucrose for 10 min at 4 °C and treated with 0.1 M glycine for 3 min at room temperature. Fixed cells were washed thrice with phosphate-buffered saline (PBS), permeabilized with 0.25% Triton X-100 for 5 min followed by washing with PBS. Cells were incubated in blocking solution (10% BSA in PBS) for 30 min at room temperature. Subsequently, the cells were incubated with primary antibodies in 3% BSA (w/v) for 1 h, washed with 3% (w/v) BSA and further incubated with secondary antibodies for 45 min at room temperature. Cells were rinsed four times with PBS for 5 min. Before dSTORM imaging, post fixation of cells was done using 2% paraformaldehyde and 2% sucrose in PBS for 10 min at 4 °C. For imaging of endogenous HCN2, cells were labeled with mouse anti-β tubulin (1 : 1000, Sigma, T8328) and rabbit anti-HCN2 (1 : 800, Alomone, APC-030) marked with Alexa 488-conjugated anti-mouse IgG (1 : 500; Invitrogen, A11029) and Alexa 647-conjugated anti-rabbit IgG (1 : 500; Invitrogen, A21245), respectively. For imaging of ectopically expressed HCN2, cells were transfected with mEos::HCN2 using Turbofect reagent prior to fixation and labelling with anti-HCN2 antibodies.