Jäderstad J, Jäderstad LM, Li J, Chintawar S, Salto C, Pandolfo M, Ourednik V, Teng YD, Sidman RL, Arenas E, Snyder EY, Herlenius E. Communication via gap junctions underlies early functional and beneficial interactions between grafted neural stem cells and the host. Proc Natl Acad Sci U S A. 2010 Mar 16;107(11):5184-9. PubMed Abstract

REAGENTS/MATERIAL:
Organotypic Cultures: To prepare mutant and wild-type mouse organoptypic cerebellar cultures, the whole cerebellum of a P9 mouse pup was removed aseptically and immersed in ice-cold dissecting medium. An anti-calbindin D-28K (Sigma Aldrich) was used to assess Purkinje neuron (PN) survival within whole-mount slices.
Immunohistochemistry: Cultures were incubated over night at 4°C in a humid chamber with the following primary antibodies: mouse monoclonal anti-nestin (Millipore); anti-GFAP (Sigma); rabbit anti-β−III-tubulin Tuj1 (Sigma); mouse monoclonal anti-MAP2ab (Millipore); mouse monoclonal anti-synaptotagmin I (Millipore); mouse monoclonal anti-connexin 26 (Invitrogen); and mouse monoclonal anti-connexin 43 (Santa Cruz); rabbit anti-connexin 26 (Invitrogen) and rabbit anti-connexin 43 (Invitrogen).
Western Blot: The filters were then immunostained using primary antibodies against Cx43 (Invitrogen). For signal detection, anti-rabbit antibodies conjugated with HRP were added, and binding was identified using enhanced chemiluminescence
Histology on Nervous (nr) Mutant Mouse: For histological evaluation primary antibodies included mouse monoclonal antibodies, mouse monoclonal anti-β-gal (Chemicon) for staining NSCs, mouse monoclonal anti-calbindin D-28K (CB-955) (Sigma Aldrich) specific for PNs and mouse monoclonal anti-NeuN (Chemicon) for GNs, and rabbit anti-connexin 43 (Chemicon) and rabbit anti-connexin 26 (Invitrogen) for gap junction proteins.