Technique of flat-mount immunostaining for mapping the olfactory epithelium and counting the olfactory sensory neurons

Abstract

The pathophysiology underlying olfactory dysfunction is still poorly understood, and more efficient biomolecular tools are necessary to explore this aspect. Immunohistochemistry (IHC) on cross sections is one of the major tools to study the olfactory epithelium (OE), but does not allow reliable counting of olfactory sensory neurons (OSNs) or cartography of the OE. In this study, we want to present an easy immunostaining technique to compensate for these defects of IHC. Using the rat model, we first validated and pre-screened the key OSN markers by IHC on cross sections of the OE. Tuj-1, OMP, DCX, PGP9.5, and N-cadherin were selected for immunostaining on flat-mounted OE because of their staining of OSN dendrites. A simple technique for immunostaining on flat-mounted septal OE was developed: fixation of the isolated septum mucosa in 0.5% paraformaldehyde (PFA) preceded by pretreatment of the rat head in 1% PFA for 1 hour. This technique allowed us to correctly reveal the olfactory areas using all the 5 selected markers on septum mucosa. By combining the mature OSN marker (OMP) and an immature OSN marker (Tuj-1), we quantified the mature (OMP+, Tuj-1-), immature (OMP-, Tuj-1+), transitory (OMP+, Tuj-1+) and total OSN density on septal OE. They were respectively 42080 ± 11820, 49384 ± 7134, 14448 ± 5865 and 105912 ± 13899 cells per mm2 (mean ± SD). Finally, the same immunostaining technique described above was performed with Tuj-1 for OE cartography on ethmoid turbinates without flat-mount.

Spatiotemporal dynamics exhibited by horizontal basal cells reveal a pro-neurogenic pathway during injury-induced olfactory epithelium regeneration

“…Hopx -expressing HBCs primarily give rise to Sus cells and olfactory sensory neurons (OSNs), 10 with the former identifiable by CK8 and interleukin-33 (IL33) located apically within the regenerated tissue 10 , 38 while immature and mature OSNs are marked by PGP9.5. 30 , 39 , 40 , 41 , 42 Following OE injury, HBC differentiation into CK8 + /apical IL33 + /tdTom + Sus cells was unaffected (17.1 HBC-derived Sus cells per 100 μm Rac1 fl/fl OE vs. 18.3 HBC-derived Sus cells per 100 μm Rac1 WT OE) ( Figures 6 A–6K). Interestingly, however, HBC-specific Rac1 cKO attenuated HBC differentiation into PGP9.5 + /tdTom + OSNs at 28 dpi by nearly half (14.6 HBC-derived OSNs per 100 μm Rac1 fl/fl OE vs. 25.7 HBC-derived OSNs per 100 μm Rac1 WT OE) ( Figures 6 L–6V).…”