Tie pfs a1r confocal microscope

Transcardial perfusions, using a 10mL syringe with a 25G winged infusion set, of 20mL 4.0% paraformaldehyde (PFA) solution were administered. Following fixative perfusion, mouse brains were removed and soaked in 4.0% PFA for 1hr. Brains were subsequently transferred to 30% sucrose solution for 24hrs and snap frozen using liquid nitrogen chilled 2-Methylbutane. Brains were embedded in optimal cutting temperature compound and mounted for cryo-sectioning (Lecia Biosystems Cryostat). 5-10μm thick sections were taken and mounted on microscope slides. Sections were dried overnight at 4°C. Tissue sections were brought to room-temperature and washed 3 times in PBS and counterstained with antifade mounting medium with DAPI (Vectashield). Brain tumor sections were imaged using an inverted Nikon TiE-PFS-A1R confocal microscope. Images were post-processed using Nikon Elements software.

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).