Methods Organotypic hippocampal slice cultures OHSC were prepared

Methods Organotypic hippocampal slice cultures OHSC were prepared from 5 to 6 day old Sprague Dawley rats according to the interface method of Stoppini et al. Pups were decapitated, the brain was removed, hippocampi were dissected and transversely sliced at a thickness of 400 Ivacaftor cystic fibrosis um, and transferred into ice cold dissection buffer containing 1% penincilin streptomicin solution, 25 mM HEPES and 10 mM TRIS in Minimum Essen tial Medium, and selected for clear hippocampal morphology. Inhibitors,Modulators,Libraries The slices were transferred onto 0. 4 um porous Millicell membrane inserts and placed in individual 35 mm plates with 1 ml of serum based medium containing 50% Minimum Essential Medium, 25% Hanks balanced salt solution, 12 mM HEPES, 25% heat inactivated horse serum Inhibitors,Modulators,Libraries and 1% penicillin streptomycin solution in a humidified chamber with 5% CO2 at 37 C.

Media was changed twice a week. All animals were cared Inhibitors,Modulators,Libraries for following procedures approved in advance by the University of Prince Edward Island Animal Care Committee, and were in accordance with the Canadian Council on Animal Care guidelines. All possible efforts were made to minimize animal Inhibitors,Modulators,Libraries suf fering and the number of animals used. DOM induced excitotoxic injury and pharmacological treatments At 13 days in vitro, damaged OHSC were excluded by propidium iodide staining using a Fluoroarc exciter lamp with a Zeiss Axioplan2 microscope. PI negative slices were ex posed to the indicated treatments. Cultures were ex posed to DOM for 24 h and then transferred to a DOM free medium.

The MEK inhibitor PD98059, the PKA inhibitor H89 as well as the CaMKII inhibitor KN93 were added to the culture medium 1 h before DOM and maintained throughout the experimental period. Immunohistochemistry Cultures were washed in 0. 1 M phosphate buffered saline, fixed in formalin for 18 h and cryoprotected in Inhibitors,Modulators,Libraries 30% sucrose PBS for an additional 18 h. OHSC were then fur ther sliced into 15 um sections on a cryostat, mounted on glass slides and stored at ? 20 C. After culturing for up to 4 weeks OHSC thin down from the original 400 um to about 180 um. For cryosectioning the first two sections of 15 um were discarded since this http://www.selleckchem.com/products/MLN-2238.html part contains the glial scar. For immunohistochemistry the next 4 5 15 um cryosections were saved which resulted in collection of the middle part of each hippocampal slice culture. The follow ing primary antibodies were used mouse anti NeuN, mouse anti GFAP, mouse anti CD11b and rabbit anti BDNF. The following secondary antibodies were used Alexa Fluor 488 goat anti rabbit IgG and Alexa Fluor 594 goat anti mouse IgG. Negative con trols for all primary and secondary antibodies were included in every run and displayed no specific staining at any time.

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