Immunostaining of iPSC-derived neurons for quantification of synaptic proteins
Erika Holzbaur, Dan Dou
Abstract
Here, we fix, permeabilize, and stain human iPSC-derived neurons for the purpose of observing and quantifying somal proteins of interest. For preceding culture of neurons, see "Protocol: Culture and transfection of iPSC-derived neurons for live-imaging of axonal cargoes."
Steps
At DIV14, fix human iNeurons in 4% paraformaldehyde supplemented with 4% sucrose for 15 minutes at 37 degrees C
Wash four times with PBS
Permeabilize for 15 minutes in 0.2% Triton-X in PBS
Block for 1 hour with 5% goat serum and 1% BSA in PBS
Incubate in primary antibody diluted in blocking solution at room termperature for 1 hour
Wash three times with PBS
Incubate in secondary antibody diluted in blocking solution for 1 hour at room temperature
Wash three times with PBS
Remove PBS and add 40 µL Prolong Gold (Thermo Fisher). Using forceps, apply coverglass.
