Rat spinal cord was processed as previously described (31 (link)) using as primary antibodies the IgGs contained in sera samples or commercial antibodies against MBP (1:1,000, Covance, Princeton, NJ, USA), NFM (1:500, Aves Lab, Tigard, Oregon, USA), GFAP (1:2,000, Dako, Glostrup, Denmark) and APC (1:300, Calbiochem, Darmstedt, Germany). To detect these antibodies we used Cy3 conjugated anti-human IgG (1:1,000, Jackson Immunoresearch, Ely, UK), Cy5 anti-chicken IgY, Alexa Fluor-594 anti-rabbit IgG (1:1,000, Invitrogen, Barcelona, Spain) and Alexa Fluor-488 anti-mouse IgG (1:1,000, Invitrogen). To detect endogenous IgG in rat spinal cord, HRP-conjugated anti-rat IgG (1:1,000; Jackson Immunoresearch) was used and their localization was detected by DAB-peroxidase reaction. Also, HRP-conjugated anti-rabbit IgG (1:1,000; Jackson Immunoresearch) was employed as a control of specificity. When performed, nuclei were counterstained with Hoescht 33,258 (1:5,000; Invitrogen).
Samples were analyzed with a LEICA SP5 confocal microscope. All post-capture image modifications were identically performed between groups, including cropping, noise reduction and minor adjustments to optimize contrast and brightness.
Samples were analyzed with a LEICA SP5 confocal microscope. All post-capture image modifications were identically performed between groups, including cropping, noise reduction and minor adjustments to optimize contrast and brightness.
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