Transpiration-assisted perfusion fixation provides in situ preservation of developing ray parenchyma cells in Eucalyptus nitens

The technique of animal vascular perfusion fixation was adapted for in situ fixation of the fragile and difficult to access cells of the ray parenchyma system in stems of 10-year-old Eucalyptus nitens trees. In situ fixation enabled tissue to be safely dissected for histological processing without risk of damage to microstructure or initiation of wound response. Fixative was perfused through the active vascular system under hydrostatic and transpiration pressure directly to vessel-associated ray cells. Diffusion from vessels allowed fixative to access nonvessel-associated ray cells. Acrolein was included to aid diffusion fixation and safranin dye was included to define fixed regions within the xylem. Sections prepared for light and electron microscopy from samples cut from regions showing intense safranin staining showed good microstructural preservation and were free of artefacts caused by mechanical injury or wound response.