Eclipse te2000 u inverted microscope

Axons growing from Xenopus eyes cultured in glass-bottom dishes were fixed with 2% paraformaldehyde (Pfa)/7.5% sucrose in 1× phosphate-buffered saline (PBS) for 20 min at room temperature (RT), washed with PBS, permeabilized with 0.1% Triton X-100 in PBS for 5 min at RT, blocked with 5% goat serum in PBS for 30 min at RT, then incubated with the primary antibody for 1 h at RT followed by a fluorochrome-conjugated secondary for 30 min at RT. Where appropriate, Phalloidin-Alexa Fluor 488 (an F-actin stain; 1 : 100, Invitrogen/Molecular Probes) was added together with the second antibody. GCs were mounted in Fluorosave (Calbiochem), coverslipped and imaged on a Nikon Eclipse TE2000-U inverted microscope fitted with an Hamamatsu C4742-80-12AG camera. Co-localization images were taken on a spinning disk microscope (UltraVIEW Vox system (PerkinElmer) mounted on an Olympus IX81 inverted microscope) equipped with an EM-CCD camera (C9100-50, Hamamatsu).

Axons cultured in glass-bottom dishes were fixed with 2% Pfa/7.5% sucrose in 1× PBS for 5 min on ice. After four quick washes with ice-cold PBS, the axons were incubated with goat anti-DCC extracellular domain antibody (R&D Systems cat. no. AF844) at 1 : 20 in PBS/0.1% BSA for 30 min on ice and washed 4× 2 min with ice-cold PBS. Axons were then fixed again with 2% Pfa/7.5% sucrose in 1× PBS for 20 min at RT, washed with PBS, permeabilized with 0.1% Triton X-100 in PBS for 5 min at RT and incubated with mouse anti-DCC intracellular domain antibody (BD Biosciences cat. no. 554223) for for 1 h at RT, followed by a mix of donkey secondary antibodies (anti-goat Alexa Fluor 568 and anti-mouse Alexa Fluor 647) for 30 min at RT. GCs were mounted in Fluorosave (Calbiochem), coverslipped and imaged on a Nikon Eclipse TE2000-U inverted microscope fitted with a Hamamatsu C4742-80-12AG camera.