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. 2017 Aug 31;7:10167. doi: 10.1038/s41598-017-10862-5

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Immunofluorescence and RNA in situ hybridization of mouse OE for activation with acetophenone and methyl salicylate. (A) Mice were stimulated by a piece of filter paper spotted with 10 µL odorant and sacrificed to stain the olfactory epithelium. pS6 and MOR161-2 were detected by Immunofluorescence and RNA in situ hybridization, respectively. (Nuclei) nuclei of olfactory sensory neurons, (MOR161-2) RNA in situ hybridization for MOR161-2, (pS6) Immunofluorescence for pS6, and (Merged) Merged image of Nuclei, MOR161-2 and pS6. And then the stained sections were observed by fluorescence microscope. Images were 20× magnification. (B) The ratio of the responding OSNs, identified using anti-S6 antibody, to the total MOR161-2 expressing cells was calculated. The means ± standard deviation of three separate experiments are shown. The statistical significance was assessed using a t test (*P < 0.05, **P < 0.01). 1% AC, 1% acetophenone; AC, acetophenone; MS, methyl salicylate; Control, purified water without odorant. PBS, with washing nasal cavity by PBS; ABT, with washing nasal cavity by PBS containing ABT. Three mice were used for each stimulation condition. At least four sections were analyzed for each stimulation condition.