Alexa 564 conjugated or 488 conjugated secondary antibodies

Hek-Na_V1.2 cells transfected with PRRT2-HA, PRRT2ΔC-HA, or PRRT2ΔN-HA constructs were live labeled by diluting primary antibodies (mouse anti-HA, 1:500, Millipore) in culture medium for 30 min at 37 °C/5% CO₂ to detect surface epitopes, followed by fixation with 4% paraformaldehyde and incubation with Alexa Fluor-594 secondary antibodies. After several washes in phosphate-buffered saline (PBS), coverslips were mounted using Prolong Gold antifade reagent (Invitrogen) containing 4′,6′-diamidino-2-phenylindole for nuclear staining. Hek-Nav1.2 cells transfected with PRRT2-HA, PRRT2ΔC-HA or PRRT2ΔN-HA constructs were fixed in 4% paraformaldehyde at room temperature for 20 min, washed in PBS, and blocked with 10% bovine serum albumin (BSA) for 20 min. Samples were sequentially incubated with mouse anti-pan-Nav (Sigma; 1:100 in 5% BSA) and rabbit anti-HA, (Invitrogen; 1:500 in 5% BSA) primary antibodies, followed by Alexa 564-conjugated or 488-conjugated secondary antibodies (Invitrogen; 1:200 in 5% BSA) at room temperature. After several washes in PBS, coverslips were mounted using Prolong Gold antifade reagent (Invitrogen) containing 4′,6′-diamidino-2-phenylindole for nuclear staining.

Neurons were fixed in 10% trichloroacetic acid (TCA; Merck) at 4 °C for 10 min, washed in PBS and blocked with 10% BSA for 20 min; samples were sequentially incubated with primary antibodies diluted in 5% BSA (α3-NKA MA3-915 monoclonal antibody from Thermo Scientific, 1:300; α1-NKA a6F monoclonal antibody (1:10, 2.7 g/mL) developed by Douglas M. Fambrough and obtained from the Developmental Studies Hybridoma Bank, University of Iowa), followed by Alexa 564-conjugated or 488-conjugated secondary antibodies (Invitrogen; 1:200) at room temperature. After several washes in PBS, coverslips were mounted using Prolong Gold antifade reagent (Invitrogen) containing DAPI (4′,6′-diamidino-2-phenylindole) for nuclear staining.