Immunohistochemical Analysis of Neural Markers

F344 × BN rats were intracardially perfused with saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer (4% PFA), while F344 frontal cortices were dissected free from fresh tissue and were fixed by immersion in 4% PFA. Tissues were cryoprotected in sucrose, frozen over liquid N₂, and sectioned coronally at 50 μm. Immunohistochemical analyses were performed using the following primary antibodies and a previously published protocol (Guadiana et al., 2013 (link)): rabbit anti-ACIII (1:10,000; Encor Biotechnology, #RPCA-ACIII), mouse anti-NeuN (1:2000; Chemicon, #MAB377), rabbit anti-Pericentrin (1:500; Covance, #PRB-432C), and goat anti-SSTR3 (1:200; Santa Cruz, #sc-11617). Appropriate species-specific, fluorophore-conjugated secondary antibodies were used to visualize binding of the primary antibodies (1:400; Jackson ImmunoResearch). To reduce lipofuscin autofluorescence, sections were treated with 0.3% Sudan Black in 70% ethanol for 10 min as previously described (Jackson et al., 2009 (link)). Stained sections were coverslipped with Prolong Antifade Gold mounting media containing DAPI (Life Technologies). Images of stained sections were captured on a Zeiss AxioObserver D1 epifluorescent microscope or an Olympus IX81-DSU spinning disc confocal microscope and are displayed as collapsed z-stacks that were collected in 1 μm steps.