Fig 1.
Summary of the 3 immunostaining techniques.
A. Table clarifying the workflow. B. Schematic presentation of the tissue preparation for observation. Ab: antibody, DAPI: 4′,6-diamidino-2-phenylindole, h: hour(s), IHC: immunohistochemistry, n.i.: not involved, OE: olfactory epithelium, OCT: Optimal cutting temperature compound, RT: Room temperature. * "Cross-section" here is a general term for the IHC technique and does not indicate the orientation of the tissue. The orientation of the nasal cavities was “coronal’ in this study. **The purpose of adhesive tape was to prevent the coverslip from sliding on the microscope slide, which could damage the sample in between.
Table 1.
List of validated primary and secondary antibodies.
Fig 2.
Workflow of the macro created for OSN counting.
The different steps of the macro created from the Fiji/imageJ software are shown in the table above. The images (A1 to A6) below are there to help better understand the table. A3: Yellow arrows indicate particle separation. A4: Red arrows indicate the particles with a surface area < 0.3μm2 that are excluded for counting. *The manual inputs include 3 steps: 1.2, 2.1 and 3.1. At completion, ‘OSNs counting’ generates a data table containing the number of green labelled OSNs, the number of red labelled OSNs and the number of colocalized OSNs (A6).
Fig 3.
Co-localization of different OSN markers with OMP, a mature OSN marker.
Immunohistochemistry (IHC) on cross sections of olfactory mucosa. The tested markers were colored in green and their name were noted on the left of each line of images. Due to the identical animal production between the anti-OMP antibody and anti-OliG2, we could not double label these two markers. The nuclei were stained in blue using DAPI. The images in the 3 left columns were acquired using an epifluorescence microscope, with a 40X objective. The images in the right column were acquired from a confocal microscope with a 60X objective.
Fig 4.
Co-localization of different OSN markers with Tuj-1, an immature OSN marker.
IHC on cross sections of olfactory mucosa. The tested markers were colored in green and their name were noted on the left of each line of images. The nuclei were stained in blue using DAPI. The images of the 3 left columns were acquired using an epifluorescence microscope, with a 40X objective. The images of the right column were acquired from a confocal microscope with a 60X objective.
Fig 5.
Immunostaining on flat-mounted septal OE using pre-selected OSN markers.
Five OSN markers (Tuj-1, OMP, DCX, N-cadherine and PGP9.5) were selected because of their staining of the OE’s surface (OSN dendrites). The co-localization with a respiratory epithelium marker, Tubulin β IV was realized on transition zones in order to show the specificity of the OSN markers. The images were acquired using a confocal microscope and presented as 3D projection. For each marker, the top pictures showed face view using a 20X objective; the bottom pictures showed a tangential view.
Fig 6.
OSN density evaluation using immunostaining of dendrites on flat-mounted septal OE.
a) Double immunostaining was done on the separated septum mucosa. The mature OSNs were stained in red using OMP and the immature OSNs were stained in green using Tuj-1. The co-stained OSN dendrites (Tuj-1+, OMP+) which appear in yellow/orange, were indicated by arrow heads. Images were acquired using a confocal microscope with a 60X objective. b) The horizontal black line illustrates the Z level of OSN where the image was acquired. c) and d) In total, 105912 ± 13899 OSN/mm2 were detected by this method: 49384 ± 7134/mm2 were immature OSNs (Tuj-1+, OMP-), 42080 ± 11820/mm2 were mature OSNs (Tuj-1-, OMP+), and 14448 ± 5865/mm2 were transitory OSNs (Tuj-1+, OMP+). The percentage in the table corresponds to the cell number ratio compared to the total number of OSNs (105912). n = 15 (3 animals and 5 different fields per animals).
Fig 7.
Direct immunostaining on ethmoid turbinates.
The morphology of whole ethmoid turbinates was visible on bright field (BF) (a), and by DAPI staining (b). The OE was highlighted by immunostaining with Tuj-1 (c and e). The merge of Tuj-1 (green) and DAPI (blue) with or without BF (gray) indicated the olfactory boundary (d and f). The photos were acquired using a fluorescence macroscope with 1.6X objective for (a-d) and 5X objective for (e, f).
Fig 8.
Summary of the localization of the different neuronal markers within the olfactory mucosa.
+: Evident staining. C: Cytoplasm. N: Nucleus. M: Membrane. Images of staining with GAP43 and Gβ, are presented in S1 Fig. * N-cadherin stained weakly olfactory fascicles.
Table 2.
Summary of the reported techniques of immunostaining on flat mounted septum mucosa.
Fig 9.
Co-labeling of OMP and Tuj-1 within the soma of the same OSN using IHC on OE cross sections.
The detection of both OMP and Tuj-1 in the cytoplasm of the same OSN was rare and the staining of the two markers was not usually superimposed. The images were zoomed from a photo acquired by a confocal microscope with a 60X objective. The arrows indicated the OSNs that co-express OMP and Tuj-1.