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. 2018 Sep 4;12:295. doi: 10.3389/fncel.2018.00295

FIGURE 7.

FIGURE 7

Identification of Cav2.1+ OSNs in the MOE. (A) Coronal MOE sections (14 μm) showing the left nasal cavity of an adult B6 mouse stained with Cav2.1. Cav2.1+ OSNs reside mainly in the MOE lining the dorsal roof (dr), nasal septum (ns), the dorsal-medial tip of endoturbinate II, and the tip of endoturbinate III (dashed lines). Few labeled OSNs are detectable at the tips of ectoturbinates 2 and 3. (B) Cav2.1 staining is present in OSN knob (k), dendrite (d), soma (s), and axon (arrowheads). To delineate axonal Cav2.1 staining, contrast and brightness was increased by 50%. (C) The peptide control reaction is devoid of Cav2.1 staining. (D) RNAscope fluorescence in situ hybridization for Cav2.1 mRNA (Cacna1a, green) in the MOE of an adult B6 mouse (coronal view, left nasal cavity) shows that the distribution of labeled OSNs is closely similar to that obtained by immunohistochemistry (A). The dorsal roof, endoturbinate I, nasal septum, and the dorsal-medial tips of endoturbinates IIa and IIb (dashed lines) show labeled OSNs. Few labeled OSNs reside at the medial tip of ectoturbinate 2 (arrowheads). (E) Higher magnification shows hybridized OSNs at all depths of the epithelial layer. (F) The negative control reaction is devoid of labeling. (G) Agarose gel electrophoresis of the products obtained by RT-PCR using Cacna1a-specific primers and total RNA of adult mouse olfactory tissue. RT-PCR resulted in products (+RT) of the expected sizes using primers amplifying exons 24–38 (1,731 bp, arrowhead) and exons 37–43 (607 bp, arrowhead). Control reactions omitting reverse transcriptase (-RT) yielded no products. M, DNA size marker in base pairs as indicated at the left. (H) Magnifications of the dorsal MOE derived at different mouse ages stained with Cav2.1. Immunoreactivity for Cav2.1 (red) is absent at embryonic day 18 (E18). Cav2.1+ OSNs become visible at about postnatal day 1 (P1), and expression continues at P7, P14, P21 toward adulthood. Images are representatives of N ≥ 2 mice per age with N ≥ 10 sections per mouse. Scale bars (A,C,D) 200 μm, (E); 50 μm; (F,H) 20 μm; (B) 10 μm.