Immunostaining of iPSC-derived neurons
Dan Dou, Erika L.F. Holzbaur
Abstract
Here, we fix, permeabilize, and stain human iPSC-derived neurons for the purpose of observing and quantifying somal and axonal abundance of proteins of interest. For preceding culture of neurons, see "Protocol: Culture and transfection of iPSC-derived neurons for live-imaging of axonal cargoes."
Steps
Replace culture media in dish with 1 mL room temperature BrainPhys culture media (Stemcell Technologies)
Add 1 mL Bouin's solution (Sigma). Incubate at room temperature for 30 minutes.
Wash at least 5 times with PBS. Yellow color should be absent.
Permeabilize in ice-cold Optima methanol (Thermo Fisher) at -20 degrees C for 8 minutes.
Wash 3 times in PBS.
Block for one hour (5% goat serum and 1% BSA in PBS).
Incubate in primary antibody in blocking solution for 1 hour at room temperature.
Wash 3 times in PBS.
Incubate in secondary antibody in blocking solution for 1 hour at room temperature.
Wash 3 times in PBS.
Remove PBS and add 40 µL Prolong Gold (Thermo Fisher). Using forceps, apply coverglass.
