Whole-Mount Immunochemical Fluorescence Confocal Microscopy
Whole tissue samples were fixed immediately after dissection, followed by 3 washes (20 min each) in PBS and further washed twice with PBS (15 min each) and treated with fresh-made sodium brohydrate (0.1%) for 10 min. They were then treated with 100% cold methanol for 5 min at -20C, followed by a 15-min rinse in PBS and another 15-min wash in PBS containing 0.1% Triton X-100. All incubations and washes were carried out under room temperature on a rocker. After blocking in 3% IgG-free BSA (Jackson ImmunoResearch, Inc) in PBS for 1 hour, samples were incubated for 2 hours in PBS containing 1% IgG-free BSA and the following primary antibodies: mouse monoclonal antibodies to neurofilament (Sigma), and bovine polyclonal antibodies to viral proteins. Following 3 washes in a large volume of PBS (~30 min each), the tissues were incubated in PBS with 1% IgG-free BSA and secondary antibodies Cy2-conjugated Donkey anti-bovine IgGs and Cy5-conjugated Donkey anti mouse IgGs for 1 hr. The double-labeled samples were washed 3 time in PBS (30 min each) and treated with 2 ug/ml PI for nuclear staining. After a 5 min rinse in PBS, samples were placed in PBS in a 60 mm petri-dish and examined using 20x or 40x water-immersion objective lenses with a BioRad MRC1024Es confocal laser scanning microscope. Series of Z-axis optical sections of the triple-labeled samples for viral proteins, nucleus, and neurofilaments were collected simultaneously with a 3-line laser excitation mode (488nm, 560nm, and 640nm) and triple emission filter set (522nm, 598nm and 680nm) using BioRad LaserSharp imaging program.