Alexa fluor 555 or 647

Following euthanasia, the pancreas of each mouse was rapidly immersion-fixed in formaldehyde, embedded in paraffin and cut in sections, rehydrated in a graded series of alcohols, washed in H₂O followed by antigen retrieval (Retrieve-ALL.1, Covance) and immunostained with mouse anti-glucagon (Abcam, 1:500) and guinea pig anti- insulin (Linco, 1:1000). The appropriate secondary antibodies were conjugated to AlexaFluor- 555 or 647 (Invitrogen). Slides were mounted with Prolong Gold (Invitrogen) and imaged by epifluorescence in an Olympus FV500 confocal microscope with 60x (NA 1.4) oil objectives. The number of alpha cells (defined as glucagon-positive) or beta cells (defined as insulin- positive) were either manually counted for each islet by a single observer in a blinded fashion and the fraction of each cell type subjected to a Kruskal-Wallis test with Dunn’s corrections for multiple testing (GraphPad Prism 6.0), or was counted by ImageJ-1.50 with the assistance of either the “cell counter plug-in” (http://rsbweb.nih.gov/ij/plugins/cell-counter.html) or with the “ITCN 1.6 plug-in” (http://rsb.info.nih.gov/ij/plugins/itcn.html) and the ratio between the cell types analyzed by Mann-Whitney test (GraphPad Prism 6.0). Cell ratios within individual islets were quantified and expressed as mean ± SE, with p-value < 0.05 considered statistically significant.

MOE sections were rinsed in 0.1 m PBS three times, 10 min each, before incubating in PBS-buffered blocking solution containing 2% normal donkey serum, 0.3% Triton X-100, and 1% bovine serum albumin for 1.5 h. Sections were then immunoreacted for 48–72 h at 4°C with primary antibodies against olfactory marker protein (OMP, 1:1000, Wako 019-22291-WAKO RRID:AB_664696) and growth-associated protein 43 (GAP43, 1:2000, Novus NB300-143 RRID:AB_10001196). After incubation with the primary antibodies, sections were washed and reacted with secondary antibodies conjugated with either Alexa Fluor 555 or 647 (1:400; Invitrogen) for 1 h at room temperature. Sections were then rinsed and coverslipped with Fluoromount-G containing DAPI, which stains nuclei (Southern Biotech). In control experiments, primary antibodies were omitted, which resulted in negative labeling.

Free-floating 250-µm hippocampal slices were permeabilized and blocked for 2 h at room temperature in a solution containing 10% normal horse serum and 0.02% Triton X-100 in PBS followed by incubation with primary antibodies in blocking buffer for 12 h at 4 °C with agitation (mouse anti-Bassoon [1:300, Novus NB120-13249], rabbit anti-Homer1 [1:300, Synaptic Systems 160002], and rabbit anti-Iba1 [1:500, Synaptic Systems 234003]). Following four 10-min washes in PBS, secondary antibodies diluted 1:1,000 in blocking buffer (donkey anti-mouse Alexa 488, donkey anti-rabbit Alexa Fluor 555 or 647; Invitrogen) were applied for 4 h at room temperature with agitation. Finally, slices were washed in PBS and mounted.