Immunocytochemistry of Neurodevelopmental Proteins

Hippocampal and iPSC derived neurons were washed 2x in PBS containing 1mM MgCl2 and 0.1 mM CaCl2 (PBS-MC). Cells were fixed for 15 minutes with a solution of 4% Paraformaldehyde/4% Sucrose in PBS-MC warmed to 37°C, washed 3x in PBS-MC, and permeabilized for 5 minutes in 0.1% Triton-X in PBS-MC. Cells were blocked in 2% BSA for an hour, then incubated in primary antibody for at least 2 hours at room temperature. Reporter protein was detected with an antibody for FLAG (mouse, Sigma) and mCherry or mApple was detected with an anti-dsRed antibody (rabbit, Clonetech). A rabbit anti-mGluR5 (Abcam) antibody was used in human neurons to verify mGluR expression. FMRP was detected with a rabbit anti-FMRP antibody (Abcam), and FMRpolyG was detected using a custom developed polyclonal rabbit antibody to the N-terminal region of the protein (NTF1, NeoScientific, 1:200, epitope: EAPLPGGVRQRGGGGGGGGGG). NTF1 staining and specificity was validated in patient derived lymphoblasts with confirmed CGG repeat sizes and loss of FMR1 mRNA expression in FXS cells (Coriell, GM09237, GM06891, GM07539) with pre-immune serum staining included as a negative control^{15 (link)}. For all staining, cells were washed 3 times, followed by incubation in secondary antibody for 1 hour. Cells were subsequently washed and placed in ProLong Gold Antifade with DAPI (Thermo Fisher).