Goat anti chicken alexa fluor 546

After 24 h in culture, media was removed and washed three times with Phosphate-Buffered Saline without Calcium and Magnesium (PBS(−/−)). Cells were fixed by adding 4% paraformaldehyde in PBS(−/−) (Cat #AAJ19943K2, Fisher Scientific) for 20 min at RT. Following this, cells were washed with PBS(−/−) three times and then blocking buffer (5% normal goat serum [Cat #PCN5000, ThermoFisher], 0.2% Triton™ X-100 [Cat #9002–93-1, Fisher Scientific], and 1% BSA [Cat #9048–46-8, Research Products International] in PBS[−/−]) was added for 30 min. Chicken polyclonal antibody to NF200 (RRID: AB_2313552, Aves) was then added (1:800 in blocking buffer) and incubated at RT for 2 hours. Culture surface was washed 3 times with PBS (−/−) followed by addition of Goat anti-Chicken Alexa Fluor®546 (RRID: AB_2534097, ThermoFisher) (1:1000) for 1 hour at RT in the dark. Following this, cells were washed with PBS (−/−) three times and then cells were mounted using Fluoromount-G® (SouthernBiotech) in the dark for 24 h before imaging.