Immunofluorescence and whole mount aorta imaging

Whole mount aorta imaging protocol

1. Harvest aorta/s from Apoe^-/- mice fed WD for 2 weeks, carefully cleaned in situ.

n.b. Aorta/s can be sectioned into several areas along its plane keeping track of the location. 

2.  Fix with 2 % Paraformaldehyde (PFA) at 4 °C for 24h. 

3. Wash with PBS and transfer to 5 ml incubation buffer [2% FBS, 0.5% saponin (cat#47036, Sigma Aldrich), 0.1 % sodium azide (cat# S2002, Sigma Aldrich)] containing Olfr2 primary antibody (1:1000 cat# OSR00013W, Thermo Fischer) and keep in agitation for 24 h at 37°C. 

4. After incubation, wash the aortas 5 times with PBS and transfer the aorta in 5 ml incubation buffer containing secondary anti-rabbit IgG-AF555 (1:500, cat# A27039 Thermo Fischer), anti-mouse CD68-AF647 (1:200,  clone FA-11, Biolegend) and Hoechst (1:10000) for 24h at 37°C in agitation. N.B. Concurrently stain also the required fluorescence minus one (FMO) controls needed.

5. After staining wash the aorta/s 5 times with PBS.

6. Using blunt curved forceps and iris scissors, open the aorta/s longitudinally, and mount it between glass slides using ProLong Glass Antifade Mountant (Cat#P36984, Invitrogen), (note: use binder clips to keep the glasses fixed and in place).

 7. Perform Imaging with a confocal scanning microscope (i.e. ZEISS LSM880). 

Note: Set image acquisition settings with control samples (i.e. unstained samples, CD68-AF647 FMO, and secondary anti-rabbit IgG-AF555 FMO) and maintain settings throughout the experiment. 

Image and scan processing can be performed with ZEN microscope software (Zeiss) and Imaris analysis software (Bitplane).