Mab354

Mice were anesthetized with an intraperitoneal injection of avertin, $0.4\mathrm{mg}/\mathrm{kg}$ , and intracardially perfused with paraformaldehyde (PFA, 4%). Brains were postfixed in PFA overnight and sliced into $75\mu \mathrm{m}$ sections. The slices were stained using a rat monoclonal Sst antibody MAB354 (Millipore) dilution 1:600, followed by a Cy3 conjugated donkey antirat antibody (1:400) and imaged using Zeiss LSM710 confocal microscope.

When necessary, depending on the tissue penetrance and antigen recognition ability of the antibody used, antigen retrieval was performed by microwaving slides in 10 mM sodium citrate buffer for 1 min at maximum power, followed by 10 min at minimum power. Slides were then washed in 1× PBS and incubated in blocking solution (5% normal donkey or normal goat serum, 0.2% Triton^® X-100 in PBS) for 1 h at room temperature. This was followed by incubation in primary antibody overnight at room temperature. Slides were washed in 1× PBS and incubated with secondary antibody solution for 1 h at room temperature. Slides were mounted in Vectashield with DAPI (Vector Laboratories). Primary antibodies used were as follows: rabbit anti-oligodendrocyte transcription factor 2 (1:300, AB9610, Millipore, Burlington, MA), rabbit anti-phosphorylated histone 3 (1:500, 06-570, Millipore), rat anti-somatostatin (1:50, MAB354, Millipore), rabbit anti-parvalbumin (1:1000, PV25, Swant, Marly, Switzerland), rabbit anti-calretinin (1:1000, Swant, 769913), and rabbit anti-Tbr1 (1:1000, gift from the Hevner laboratory, University of Washington School of Medicine, Seattle, WA). The following secondary antibodies were used: (1:250 dilution, Thermo Fisher Scientific): donkey anti-rabbit 555 (A31572), goat anti-rabbit 546 (A11035), goat anti-rabbit 488 (A11008) and goat anti-rat 488 (A11006).

For immunocytochemistry, single cells were fixed in 2.5% paraformaldehyde and kept at 4 °C. On the day of the experiment, cells were permeabilised with 0.1% Triton X-100 in phosphate-buffered saline (PBS) for 5 min at room temperature. Permeabilisation was followed by blocking of nonspecific binding with PBS containing 5% swine serum for 20 min. The cells were then incubated overnight with anti-SGLT2 antibodies (1:75, Santa Cruz Sc-393350) at 4 °C. On day 2, cells were incubated with mouse Alexa-488 Tyramide kit (T20948, Molecular Probes) according to manufacturer’s instruction. Cells were then incubated in primary antibodies anti-somatostatin (1:100, Millipore, MAB354) for 1 h at room temperature, followed by secondary antibodies Alexa 568 anti-rat (1:500, A-11077, Invitrogen) for 30 min by the nuclear stain RedDot2 (1:200, 40061, Biotium) for 10 min at room temperature. Samples were visualised with a Bio-Rad confocal microscope using appropriate lasers and filters.

Following terminal general anesthesia, mice were transcardially perfused with 4% PFA in PBS and postfixed for 1–2 hr depending on age. Brains were washed in PBS, cryoprotected by exposure to 10% then 30% sucrose in PBS before being embedded in O.C.T. (VWR) on dry ice. Tissues were cryosectioned at 14–16 μm and mounted on slides. Prior to immunohistochemistry, slides were washed with PBS, then PBST (0.1M PBS, 0.1% Triton X-100) and blocked with 2% donkey serum in PBST for 1 hr at RT. Slides were incubated with primary antibody (Ab) in blocking solution overnight at 4°C. The following Abs were used: rabbit anti-Lhx6 (1:400; gift from V Pachnis) rabbit anti-SST (AB5494, Millipore), rat anti-SST (MAB354, Millipore), mouse anti-PV (MAB1572, Millipore), and chicken anti-GFP (ab13970, Abcam). Prior to incubation with the relevant secondary Ab (1:200; fluorophores: Cy2, Alexa488, Cy3, Alexa 546, Cy5, AMCA; Abcam/Millipore), slices were washed thoroughly in PBS for 2 hr at RT. Sections were washed and counterstained with DAPI before being mounted and sealed using nail polish. Images were acquired using a Zeiss laser scanning confocal microscope (LSM710).

Mice were fixed by transcardiac perfusion with 4% paraformaldehyde and brains were post-fixed by overnight immersion in 4% paraformaldehyde at 4°C. Samples were cryoprotected in 30% sucrose and frozen. Cryoprotected brains were embedded in OCT (Leica, France) and 60-μm-thick free-floating cryostat sections were prepared.
For lacZ expression analysis, adult brains were fixed by perfusion with 4% PFA with no postfixation. X-gal staining was performed as described [18 (link)]. Immunohistochemistry on tissue sections was performed on free-floating sections (60 μm) of adult Dlx5^lacZ/+ brains, incubated overnight at 4°C with a chicken anti β-D-galactosidase antibody (1:2000; Aves labs BGL-1040) combined with either mouse anti PV (1:2000, Sigma P3088), rabbit anti Calretinin (1:1000, Millipore AB5054) or rat anti Somatostatin (1:1000, Millipore MAB354). Sections were then incubated for 2 hours at room temperature in the corresponding secondary fluorescent antibodies (1:300; Jackson Immunoresearch). Pictures were acquired using a Leica SP5 confocal microscope.