Mouse monoclonal anti v5 tag

Coverslips were fixed in PBS with 4% methanol-free formaldehyde
(#PI28906, Fisher Scientific) for 15 mins, washed three times with TBS,
permeabilized with TBS containing .05% Triton X-100 (#BP151–100,
Thermo Fisher Scientific), washed three times with TBS, and blocked with TBS
containing .02% Triton X-100 and 5–10% donkey serum
(#S30–100ML, Millipore Sigma). Coverslips were incubated with
antibodies and dilutions specific to each experiment in TBS containing .02%
Triton X-100 and 5% donkey serum overnight at 4°C. Antibodies and
dilutions used are as follows: goat-polyclonal anti-FOXP2 (N-16),
(#sc-21069, Santa Cruz Biotechnology, Dallas, Texas), 1:1000;
mouse-monoclonal anti-TUJ1 (#801201, BioLegend, San Diego, California),
1:1000; rabbit-polyclonal anti-NFIA (#HPA008884, Millipore Sigma), 1:500;
rabbit-polyclonal anti-NFIB (#HPA003956, Millipore Sigma), 1:500;
mouse-monoclonal anti-V5-tag (#R960–25,Thermo Fisher Scientific),
1:10,000. This was followed by incubation with 1:10,000 of the appropriate
Alexa Fluor® IgG secondary antibodies (Thermo Fisher Scientific) in
TBS containing .02% Triton X-100 and 5% donkey serum. Coverslips were
mounted with ProLong® Diamond Antifade Reagent with DAPI (P36962,
Thermo Fisher Scientific) and imaged using a Zeiss Observer.Z1 inverted
microscope and ZEN 2011 software.

Proximity ligation was performed using the Duolink PLA Fluorescence
reagents (#DUO92101, Millipore Sigma) as per the manufacturers’
instructions. Samples were probed with mouse-monoclonal anti-V5-tag
(#R960–25, Thermo Fisher Scientific) at a 1:10,000 dilution and
either rabbit-polyclonal anti-NFIA (#HPA008884, Millipore Sigma) at a
1:10,000 dilution or rabbit-polyclonal anti-NFIB (#HPA003956, Millipore
Sigma) at a 1:10,000 dilution. Coverslips were imaged using a 60x objective
on a Zeiss Observer.Z1 inverted microscope using ZEN 2011 software. The
level of exposure in the red, PLA signal channel was kept consistent across
conditions. Using FIJI, brightness and contrast were auto adjusted in the
DAPI channel for visualization. Background was subtracted and brightness was
adjusted in the red, PLA signal channel in order to visualize puncta above
background signal.