The samples were fixed with 4% paraformaldehyde
(PFA) for 1 h at room temperature (RT) and then permeabilized with
0.1% PBST (0.1 M PBS with 0.01% Triton X-100). Specifically, for PSD95
staining, the samples should be incubated with 0.1× pepsin (DAKO,
S3002) for 3–5 min for antigenic repair. This step was not
necessary for other antigen stains. The cells were blocked with a
blocking solution for 1 h at RT, then the solution was replaced with
the corresponding primary antibody at 4 °C overnight. The primary
antibodies used in this work are as follows: anti-PSD95 (Millipore,
MAB1596), anti-Tuj1 antibody (Abcam, ab78078), and anti-Synapsin-1
antibody (Cell Signaling Technology, 5297T). The cells were washed
three times with 0.1% PBST and then incubated with the corresponding
secondary antibodies and DAPI for 1 h at RT. The secondary antibodies
included donkey antirabbit 555 (Invitrogen, A31572), donkey antimouse
555 (Invitrogen, A31570), goat antimouse IgG2a 488 (Invitrogen, A21131),
and Alexa fluor 488 phalloidin (Thermo Fisher Scientific, A12379).
Finally, the antifluorescence quencher DAKO (DAKO, S3023) was used
to cover the slides. A Zeiss LSM900 confocal microscope was used to
observe and capture images.
(PFA) for 1 h at room temperature (RT) and then permeabilized with
0.1% PBST (0.1 M PBS with 0.01% Triton X-100). Specifically, for PSD95
staining, the samples should be incubated with 0.1× pepsin (DAKO,
S3002) for 3–5 min for antigenic repair. This step was not
necessary for other antigen stains. The cells were blocked with a
blocking solution for 1 h at RT, then the solution was replaced with
the corresponding primary antibody at 4 °C overnight. The primary
antibodies used in this work are as follows: anti-PSD95 (Millipore,
MAB1596), anti-Tuj1 antibody (Abcam, ab78078), and anti-Synapsin-1
antibody (Cell Signaling Technology, 5297T). The cells were washed
three times with 0.1% PBST and then incubated with the corresponding
secondary antibodies and DAPI for 1 h at RT. The secondary antibodies
included donkey antirabbit 555 (Invitrogen, A31572), donkey antimouse
555 (Invitrogen, A31570), goat antimouse IgG2a 488 (Invitrogen, A21131),
and Alexa fluor 488 phalloidin (Thermo Fisher Scientific, A12379).
Finally, the antifluorescence quencher DAKO (DAKO, S3023) was used
to cover the slides. A Zeiss LSM900 confocal microscope was used to
observe and capture images.