Explant cultures were fixed in 4% paraformaldehyde
(EMS, Hatfield, PA) and permeabilized in 100% ice-cold methanol (Fisher
Scientific, Hampton, NH). After blocking with 5% normal goat serum,
samples were incubated with anti-MBP antibodies, overnight at 4 °C.
Bound antibodies were detected with Alexa Fluor 488 goat antirat IgG
(Molecular Probes, Eugene, OR). Coverslips were mounted using the
Prolong Antifade kit (Molecular Probes). Proximal regions of sciatic
nerves were sectioned (5 μm thickness) and processed for immunostaining
with anti-PMP22 antibodies, as described.13 (link) AlexaFluor 594-conjugated goat antirabbit antibodies were used to
detect the bound primary antibodies. Samples which were processed
in parallel without incubation with primary antibodies served as the
negative controls. Images were obtained using a SPOT digital camera
(Diagnostic Instrumentals, Sterling Heights, MI), with a Nikon Eclipse
E800 or an Olympus DSU spinning disc confocal (Tokyo, Japan) microscope,
using identical exposure settings. Images were processed using Photoshop
(Adobe Systems).
(EMS, Hatfield, PA) and permeabilized in 100% ice-cold methanol (Fisher
Scientific, Hampton, NH). After blocking with 5% normal goat serum,
samples were incubated with anti-MBP antibodies, overnight at 4 °C.
Bound antibodies were detected with Alexa Fluor 488 goat antirat IgG
(Molecular Probes, Eugene, OR). Coverslips were mounted using the
Prolong Antifade kit (Molecular Probes). Proximal regions of sciatic
nerves were sectioned (5 μm thickness) and processed for immunostaining
with anti-PMP22 antibodies, as described.13 (link) AlexaFluor 594-conjugated goat antirabbit antibodies were used to
detect the bound primary antibodies. Samples which were processed
in parallel without incubation with primary antibodies served as the
negative controls. Images were obtained using a SPOT digital camera
(Diagnostic Instrumentals, Sterling Heights, MI), with a Nikon Eclipse
E800 or an Olympus DSU spinning disc confocal (Tokyo, Japan) microscope,
using identical exposure settings. Images were processed using Photoshop
(Adobe Systems).
