For HeV antigen localisation in brain cell studies, animals were deeply anesthetised and euthanased by perfusion with paraformaldehyde to achieve optimal fixation of tissues for confocal microscopic studies. The thoracic cavity was opened and the right auricle of the heart removed. Three ml saline was injected into the left ventricle of the heart to achieve exsanguination. Following saline injection, 11 ml of 4% paraformaldehyde (volume/volume) was slowly injected into the left ventricle of the heart, perfusing the entire animal. Following perfusion, mice were placed under 4% paraformaldehyde soaked tissues and allowed to fix for 1 hour. After initial fixation, the brain was removed and immersion fixed in 4% paraformaldehyde (volume/volume) for a further 24 hours after which it was placed into PBSA for processing.