In situ proximity ligation assay and analysis of neurite length
To study the GALR2-Y1R heteroreceptor complexes, the in situproximity ligation assay (in situ PLA) was performed as described
previously (Borroto-Escuela et al., 2021;
Narváez et al., 2021). After
permeabilization with PBS containing 0.2% Triton X-100 for 5 min, cells
were treated with PBS containing 1% bovine serum albumin. The
hippocampal cells were then incubated with the primary antibodies
diluted in a suitable concentration in the blocking solution at 4 °C
overnight. Then, cells were washed twice, and the proximity probe
mixture (Duolink PLA probe anti-goat MINUS and Duolink PLA probe
anti-rabbit PLUS, Sigma-Aldrich, Stockholm, Sweden) was applied to the
samples and incubated for 1 h at 37 °C in a humidity chamber. The
unbound proximity probes were removed by washing the slides twice, 5 min
each time, with blocking solution at room temperature under gentle
agitation and the sections were incubated with the
hybridization-ligation solution (BSA, 250 g/ml), T4 DNA ligase (final
concentration of 0.05 U/μl), 0.05% Tween-20, 250 mM NaCl, 1 mM ATP and
the circularization or connector oligonucleotides (125–250 nM)) and
incubated in a humidity chamber at 37 °C for 30 min. The excess of
connector oligonucleotides was removed by washing twice, for 5 min each,
with the washing buffer A (Sigma-Aldrich, Duolink Buffer A (8.8 g NaCl,
1.2 g Tris Base, 0.5 ml Tween 20 dissolved in 800 ml high purity water,
pH to 7.4) at room temperature under gentle agitation and the rolling
circle amplification mixture (Duolink amplification red, DUO82011,
Sigma-Aldrich, Stockholm, Sweden) was added to the cells and incubated
in a humidity chamber at 37 °C for 100 min. Then, the cells were
incubated with the detection solution in a humidity chamber at 37 °C for
30 min. In a last step, the cells were washed twice in the dark, for 10
min each, with the washing buffer B (Sigma-Aldrich, Duolink Buffer B
(5.84 g NaCl, 4.24 g Tris Base, 26.0 g Tris-HCl. Dissolved in 500 ml
high purity water, pH 7.5) at room temperature under gentle agitation.
The coverslips were put on a microscope slide and a drop of appropriate
mounting medium (e.g., Duolink Mounting Medium, Sigma-Aldrich) was
applied and sealed with nail polish. The slides were protected against
light and stored for several days at −20 °C before confocal microscope
analysis. The in situ PLA experiments were performed using the following
primary antibodies: rabbit anti GALR2 (Alomone Lab, 1:100) and goat anti
NPYY1R (sc-21992 Santa Cruz Biotechnology INC, CA, 1:200). Furthermore,
cells were labelled with Neuro-Chrom Pan Neuronal Marker primary
antibody (ABN2300, 1:100, Sigma-Aldrich; Merck Life Science S.L.U.) for
1 h, extensively washed, and stained with the green fluorescence
secondary antibody goat anti-rabbit DyLight 488 (Jackson Laboratories
InmunoResearch, 1:100). Cell nuclei were counterstained with DAPI (blue)
contained in the mounting medium. The negative control consists in the
omission of the species-specific primary antibody corresponding to the
GALR2 in the presence of the two PLA probes. As a positive control of
the PLA assay, a parallel analysis of the 5-HTR1A-5HTR2A isoreceptor
complexes has been performed as previously documented
(Borroto-Escuela et al., 2017).
Acquisition of microscopy images, In situ PLA data analysis and
morphometric quantifications were performed as previously described
(Narvaez et al., 2020).