Ix 81 dsu spinning disc confocal microscope

Cell cultures were fixed with 4% paraformaldehyde prepared in 1 × CBS (Posada-Duque et al., 2017 (link)). Autofluorescence was eliminated using 50 mM NH₄Cl. The cells were permeabilized with 0.2% Triton X-100 prepared in 1 × CBS and subsequently blocked with blocking solution (2.5% FBS in 1 × CBS). The culture plates were incubated overnight at 4°C with primary mouse antibodies against MAP2 (1:750, Sigma-Aldrich, 2-G3893) to stain neurons, GFAP (1:750, Sigma-Aldrich, 3-M4403), s100β (Dako, IS504) and GS (1:500, Molecular Probes; 701989) to stain astrocytes, and IBA-1 (1:500, Wako, 019-19741) to stain microglia, and p120 ctn (1:1000, Sigma-Aldrich, 15D2) to stain endothelium. Subsequently, the samples were incubated with secondary Alexa 594 or 488 antibodies (1:500, Molecular Probes), Hoechst 33258 (1:5,000, Invitrogen) and phalloidin conjugated to Alexa 488 or 594 (1:500, Molecular Probes). The coverslips were mounted onto slides with FluorSave (Calbiochem). Observation and image capture of cells were performed using an Olympus IX 81 epifluorescence microscope with 20 × objective (NA, 0.5) and 60 × oil immersion objective (NA 1.42) lenses. The dendritic spines in neurons were imaged using an Olympus IX 81 DSU spinning disc confocal microscope with a 60 × objective lens (NA, 1.42) with immersion oil.

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.