Coreldraw v 18

Analysis and graphical presentation of the data were performed with Imaging Workbench 6 (INDEC), Microsoft Excel, Clampfit 9.2 (Molecular Devices), NIH ImageJ 1.52 (http://imagej.nih.gov/ij) and assembled in CorelDraw v.18 (Corel). Statistical analysis was performed in Microsoft Excel and Graph Pad Prism 8.

Mice were killed by inhalation of carbon dioxide followed by cervical dislocation. Freshly dissected turbinates and septum were drop fixed for 3–4 h on ice in freshly prepared 4% paraformaldehyde in a phosphate-buffered saline (PBS), pH 7.4 supplemented with 20% sucrose. Tubes containing the tissue were carefully placed in a refrigerator at 4°C and left for the duration of fixation without any movement or agitation. This step was critical for the preservation of cilia, which are known to be extremely sensitive to mechanical damage. The tissue was thoroughly washed in PBS and blocked with PBS containing 3% fetal bovine serum, 2% bovine serum albumin and 0.3% Triton X-100 for 2 h at room temperature. The tissue was then incubated with primary antibody against mouse M71/72 olfactory receptor (a gift from Dr Gilad Barnea, Brown University, Providence, USA) raised in guinea pig, diluted 1:1000 in the same blocking solution. Finally, the tissue was incubated with secondary anti-guinea pig-IgG conjugated to Alexa Fluor 568 (1:1000) for 2 h and placed in antifading mounting agent Vectashield (Vector Labs) on the glass coverslip. Specimens were analyzed in an inverted Nikon TiE-PFS-A1R confocal microscope. Images were post-processed using Nikon Elements software (version 4.30) and NIH ImageJ (Wayne Rasband, NIH, http://imagej.nih.gov/ij) and assembled in CorelDraw v.18 (Corel).