Quantification of Olfactory Neuron Markers

As previously described (Ueha et al., 2014 (link), 2016a (link)), all samples were cut at the level of the anterior end of the olfactory bulb. Four-micrometer thick paraffin sections were deparaffinized in xylene and rehydrated in ethanol before immunostaining. For immunostaining, deparaffinized sections were treated with 3% hydrogen peroxide to block endogenous peroxidase activity and were incubated in Blocking One (Nacalai Tesque, Kyoto, Japan) to block non-specific immunoglobulin binding. Primary antibodies were detected using peroxidase-conjugated secondary antibodies and a diaminobenzidine (DAB) substrate. Three different microscope fields (dorsal, middle and ventral) of each bilateral septal OE were captured using a digital microscope camera (Keyence, Osaka, Japan, BZ-9000) with a 40× objective lens (Figure 1B). Analyses were restricted to the OE of the nasal septum to minimize variation between specimens (Figure 1C). The numbers of olfactory marker protein-positive (OMP⁺) ORNs was quantified by averaging the number of cells in each of the three microscopic fields between sections. The number of SOX2⁺ ORN progenitors, GAP43⁺ immature ORNs, Ki67⁺ cells, and cleaved Cas3⁺ apoptotic cells per mm of basal layer length were manually counted using digital imaging software (Photoshop CS6 Adobe, San Jose, CA, USA), in a blinded manner for each of the three different fields.