Anti parvalbumin

To assess the activation of PV⁺ interneurons in the PrL and IL regions during natural aggression, mice were perfused with saline and 4% paraformaldehyde under deep anesthesia. The brains were fixed overnight at 4°C, and then the fixative was replaced with 30% sucrose solution for dehydration until the brains sank to the bottom of the container. The brains were cut into 30-μm-thick coronal sections with a freezing microtome (CryoStar NX50, Thermo, USA). Sections were washed in PBS three times (5 min each time) and permeabilized with PBS containing 0.5% Triton X-100 at 37°C for 30 min. Then, sections were blocked in 4% goat serum for 2 h at room temperature. The sections were incubated with the primary antibody at 4°C overnight (anti-parvalbumin, 1:500 concentration, Millipore, MAB1572, USA; c-Fos, 1:500 concentration, CST, #2250, USA). After three washes with PBS for 5 min each time, sections were incubated with species-specific fluorophore-conjugated secondary antibodies (Invitrogen A-11029 and A-21428) for 2 h at room temperature. Finally, DAPI was used to counterstain the nuclei after three washes in PBS. Images were acquired to verify virus expression and cell numbers. For c-Fos immunofluorescent staining, a total number of 16 C57Bl/6 mice (n = 4 from each group) were included.

Immunostaining of brain sections was performed as described previously^{61 (link),62 (link)}. The following primary antibodies were used: anti-Parvalbumin (Millipore, Rabbit, AB1572; 1:200), anti-Arid1b (Abcam, Mouse, ab57461; 1:200), anti-VIAAT (PhosphoSolutions, Rabbit, 2100-VGAT, 1:500), anti-VGLUT1 (Millipore, Guinea pig, AB5905, 1:1000), GAD2 (Developmental Studies Hybridoma Bank, Rat, GAD6, 1:200), and anti-Somatostatin (MilliporeSigma, Rat, MAB354; 1:200). 4′,6-diamidino-2-phenylindole (DAPI; Sigma; 1μg/ml) was used for counterstaining. Appropriate secondary antibodies conjugated with Alexa Fluor dyes (Invitrogen) were used to detect primary antibodies. All slides were visualized and imaged under FV3000 (Olympus) fluorescent confocal microscope system. To quantify ARID1B expression levels, we measured the intensity of ARID1B fluorescence in PV or SST positive cells using NIH ImageJ. The expression intensities were averaged and presented after background subtraction and normalization to those of PV or SST negative cells.

Sections were immunolabeled as previously described (Ellisor et al., 2009 (link)). Sagittal sections from Wnt1-Venus embryos at E8.5, E10.5, and E12.5 were analyzed using an anti-GFP antibody (1:600, Molecular Probes, Cat # A-6455). Adult coronal sections for fate mapping experiments were immunolabeled with an anti-β-galactosidase (β-gal) antibody (1:500, Biogenesis, Cat # 4600-1409 or 1:500, Abcam, Catalog # ab9361-250) to identify Wnt1-derived cells (Ellisor et al., 2009 (link)). In addition, anti-Calretinin (1:5000, Chemicon; Billerica, MA; Catalog # AB1550), anti-Calbindin (1:1000, Swant, Catalog # CB3a), and anti-Parvalbumin (1:1000, Sigma, Catalog # P3088-.2ML) antibodies were used as biomarkers. Secondary antibodies were prepared at 1:500 and include: Alexa 488 (Invitrogen; Cat # A-21206, donkey anti-rabbit IgG; Cat # A-21202, donkey anti-mouse IgG; Cat #A-11055 donkey anti-goat IgG), Dylight 549 (Jackson ImmunoResearch Laboratories; Cat #703-505-155, donkey anti-chicken).