Gey s balanced salt solution

Nasal pits of E11.5 GnRH-GFP mice were isolated under aseptic conditions in Gey's Balanced Salt Solution (Invitrogen) enriched with glucose (Sigma-Aldrich). Nasal explants were placed onto glass cover slips coated with 10 ml of chicken plasma (Cocalico Biologicals, Inc.). Thrombin (10 ml; Sigma-Aldrich) was then added to allow the explant to adhere to the cover slip (Thrombin/plasma clot). Explants were maintained in defined serum-free medium containing 2.5 mg ml⁻¹ fungizone (Sigma-Aldrich) at 37 °C with 5% CO₂ for up to 30 days in vitro. From culture day 3 to day 6, fresh media containing fluorodeoxyuridine (8 × 10⁻⁵ M; Sigma-Aldrich) was provided to inhibit the proliferation of olfactory neurons and non-neuronal tissue. The medium was replaced by fresh serum free medium (SFM) twice a week. Nasal explants were fixed for 1 h at 7 days in vitro with 4% PFA and processed for immunocytochemistry.

Mouse pups between 8 and 11 day old were decapitated, and the NG were quickly removed and cultured in chilled Gey's Balanced Salt Solution (Invitrogen) enriched with glucose (0.5%) and KCl (30 mM). The NG were then placed on Millicell-CM filters (Millipore; pore size 0.4 μm) and then maintained at the air-media interface in minimum essential medium (Invitrogen) supplemented with heat-inactivated horse serum (25%, Invitrogen), glucose (32 mM), and GlutaMAX (2 mM, Invitrogen). Cultures were typically maintained for 10 days in standard medium, which was replaced three times a week. After an overnight incubation in low serum, (1.5%) MEM supplemented with GlutaMAX (2 mM), slices were stimulated with vehicle, 5 μM GW3965 for 4 hr. RNA was harvested using Acturus PicoPure RNA Extraction kit (Applied Biosystems).

Organotypic cultures were prepared as previously described (Askvig et al. 2013 (link)). Briefly, 6-day-old Sprague Dawley rat pups were decapitated and their brains were removed and placed in chilled Geys Balanced Salt Solution (Gibco, Grand Island, NY) enriched with glucose (5 mg/ml; Sigma). The brains were then trimmed to remove exterior cortical material and 350 μm coronal sections obtained using a McIlwain Tissue Chopper (Stoelting). The sections containing the magnocellular neurosecretory system nuclei were placed in chilled Geys Balanced Salt Solution and then trimmed dorsal to the third ventricle and lateral to the SON under a dissecting microscope. Sections from each animal were then placed on a single Millicell-CM filter insert (pore size 0.4 μm, 30 mm diameter; Millipore, Bedford, MA) and each filter insert was then placed in a 35 × 10 mm Petri dish containing 1.1–1.2 ml of culture media for the experimental period.

Striatal slice cultures were prepared as previously described with a few modifications [40] (link). In brief, newborn C57BL/6 mice (postnatal day 0–1) were quickly decapitated and the brains isolated under aseptic conditions. After removal of the meninges, brains were placed on their ventral surfaces and cut into 400 µm coronal slices using a McIlwain tissue chopper (Mickle laboratory, Cambridge, UK). The tissue was immediately transferred to Gey's Balanced Salt Solution (Gibco) supplemented with 5% glucose (Merck) for subsequent slice separation and removal of neocortical and subventricular tissue. Striatal slices were placed on semiporous membranes (pore size 0.4 µm, Millipore, Bedford, MA, USA; 4 slices/membrane) and placed as inserts in six-well culture trays with 1 mL serum-containing culture medium per well. The medium was composed of 25% heat-inactivated horse serum (Gibco), 25% Hanks balanced salt solution (HBSS; Gibco), 25 mmol/L -glucose (Merck), 1% penicillin/streptomycin in OPTIMEM (Gibco). The slice cultures were grown at either high (20%) or low (3%) oxygen tension.

Organotypic brain slice cultures were prepared and grown by the interface culture method as also previously described35 (link)36 (link). In brief, postnatal day 0–1 mice were quickly decapitated and the brains isolated under aseptic conditions. After removal of the meninges and isolation of the brain stem (midbrain and pons, excluding medulla oblongata), tissue blocks were sectioned transversely at 400 μm using a McIlwain tissue chopper (Mickle Laboratory). The slices were immediately transferred to chilled Gey's balanced salt solution (Gibco) supplemented with 30 mmol l⁻¹ glucose and 30 mmol l⁻¹ KCl (Merck) for subsequent separation and trimming of the slices. Three tissue slices from the same brain were placed on each semiporous membrane insert (pore size 0.4 μm; Millipore, Bedford, MA, USA) and transferred to six-well culture trays with 1 ml serum-containing culture medium per well. The medium was composed of 25% heat-inactivated horse serum (Gibco), 25% Hanks balanced salt solution (Gibco), 25 mmol l⁻¹D-glucose (Merck) in OPTI-MEM (Gibco). The organotypic brain slice cultures were grown at 36 °C in 5% CO₂ for 48 h before HSV-1 infection (Mckrae; 5 × 10³PFU in 3,5μl per brain slice). Two days post infection culture medium and six days post infection, brain slices were collected, homogenized and the viral loads determined as described above.