Mab377

Immunofluorescence and immunochemistry were performed as previously described (Cosset et al., 2015 (link), 2016 (link)). The following primary antibodies were used: mouse anti-neuronal nuclei-specific protein (NeuN) (Chemicon;MAB377), rabbit anti-βIII-tubulin (Covance;PRB435P), goat anti-ChaT (Chemicon; AB144P), rabbit anti-HB-9 (Abcam; ab922606), rabbit anti-ISLET1 (Abcam; ab22450), goat anti-GALR3 (Abcam), mouse anti-EV-71 (Abcam; ab36367), and mouse anti-PV-3 (Abcam; ab22450). Alexa Fluor (555 and 488)-labeled antibodies from goat or donkey against mouse, goat, or rabbit (Molecular Probes) were used as secondary antibodies. Cell nuclei were stained with DAPI (4, 6-diamidino-2-phenylindole). For IHC, biotin-conjugated anti-rabbit IgG or anti-goat IgG were used and developed using avidin-biotin peroxidase detection system (Vector Labs) with 3,3′-diaminobenzidine substrate (DAB, Sigma-Aldrich) after 2 min of incubation.

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.