Fluorescent staining was performed for 30 min at room temperature using highly cross-absorbed Alexa Fluor 647 secondary antibodies (Life Technologies).
All animal specific connexin antibodies used highly cross-react with the relevant human connexins [ 29].
Rabbit polyclonal anti-OGFr primary antibody (Proetintech, IL, USA) was used at a dilution of 1 25 and the secondary antibody (anti-Rabbit, highly cross-adsorbed IgG; Invitrogen, USA) was used at a dilution of 1 200.
Incubation with the secondary antibody was performed for 30 min at room temperature using anti-rabbit or anti-mouse IgG (Alexa Fluor 546 highly cross-adsorbed, diluted 1∶300, Invitrogen).
To exclude cross-reactivity of secondary antibodies during co-labelling experiments, batches of highly cross-adsorbed secondary antibodies were routinely tested using antibodies against distinct targets, that is, a rabbit-anti-EEA1 antibody (CST, #3288) or a rat anti-Lamp antibody (Santa Cruz, Cat# sc19992) and mouse anti-PrP antibody (ICSM18), followed by incubation with secondary antibodies.
Highly cross-adsorbed Alexa Fluor anti-mouse 488 or anti-rabbit 594 secondary antibodies were used for detection (Invitrogen).
Secondary antibodies (donkey, highly cross-adsorbed) were AlexaFluor®680- or IRDye®800-labeled (Invitrogen Corporation, Carlsbad, USA and Rockland Immunochemicals Inc., Gilbertsville, USA respectively).
Following three rinses in 0.1 M PBS containing 0.05%TWEEN, the cells were incubated with a highly cross-adsorbed, fluorophore-conjugated secondary antibody (AlexaFluor 488 or 594, Molecular Probes, Carlsbad, CA) for two hours at room temperature.
Sections were then incubated overnight in mouse monoclonal anti-NeuN (1∶500; Chemicon), in Alexa-488 goat anti-mouse highly cross-adsorbed antiserum (1∶200; Molecular Probes) and counterstained with DNA dye bisbenzimid (Hoechst 33342, Sigma; 1 ug/mL).
Detection was achieved by highly cross-adsorbed goat anti-guinea pig Alexa-647 and highly cross-adsorbed goat anti-rat Alexa-488 (Invitrogen, Carlsbad, CA, USA).
