Anti-Neurexin-3-alpha [IPI-mNRXN3a.67] in immunohistochemistry (IHC) (Mouse)

Animals were anaesthetised by intraperitoneal injection of 0.1 mL pentobarbital sodium BP 20% (Dolethal, Vetoquinol UK) and transcardially perfused with 10 mL phosphate buffered saline (PBS, Oxoid), followed by 10 mL 4% paraformaldehyde (Alfa Aesar 16%, diluted 1:4 in PBS). The brains were dissected and immediately post-fixed in same fixative for 3–4 h at 4 °C. Fixed samples were cryoprotected by incubating in 5 mL 30% sucrose (VWR International) in PBS for a minimum of 48 h at 4 °C or until they sank. Brains were frozen in Optimal Cutting Temperature (OCT, CellPath) using a beaker containing isopentane (VWR Chemicals) submerged into liquid nitrogen and stored at -80 °C.

Frozen mouse brain sections were defrosted to room temperature and rinsed with Tris buffer solution (1X TBS, pH 7.4, Sigma) for 5 min before addition of blocking solution containing 5% bovine serum albumin (BSA, Sigma) and 0.5% Triton X-100 (Sigma) in 1X TBS. Slides were incubated at room temperature for 2 h in 100μL blocking solution prior to overnight incubation at 4 °C in 100 μL primary antibody and diluent solution (3% BSA, 0.2% Triton X-100 in 1X TBS). Sections were then washed three times for 10 min each before secondary antibody incubation. The washing step was repeated, followed by mounting with a glass coverslip (VWR, 1.5 thickness, 18 mm diameter) using Mowiol mounting reagent (Sigma-Aldrich) with 1,4-diazabicyclo [2.2.2]octane (DABCO). Slides were left to curate overnight in a dry staining chamber before imaging.

Sagittal tissue sections (18 μm thickness) were cut using a Leica CM3050S cryostat at -20 °C. PBS was used as adhesive to collect tissue onto SuperFrost Plus slides (Thermo Scientific, J1800AMNZ). Slides were left to dry overnight at room temperature and stored at -20 °C until use.