From the experiments described above, brain tissue harvested 3 dpi from 1-day-old chicks infected by intracerebral route and various tissue samples harvested 4 dpi from 2-week-old chickens infected by the intranasal route were fixed in phosphate-buffered formalin (10%). Fixed tissues were embedded in paraffin and sectioned (Histoserv, Inc., Germantown, MD). Sections from mock-infected birds were used as controls. The tissues were deparaffinized, rehydrated, and subsequently, immunostained to detect viral N protein using the following protocol. Briefly, the sections were blocked with 1% BSA in PBS for 1 h at room temperature, incubated with a polyclonal antibody (1:200 dilution) against the respective N protein (29, 30) followed by horseradish peroxidase-conjugated goat anti-rabbit antibodies for 30 min, and then stained with AEC (3-amino-9-ethylcarbazole) substratechromogen.