Fixation methods for electron microscopy of human and other liver

Figure 1 Diagram schematically depicting the different and most frequently used perfusion-fixation methods, including perfusion-fixation needles, to fix hepatic tissue. A: Gravity-mediated perfusion fixation using 12 cm water pressure: (1) pre-perfusion buffer; (2) fixative solution; (3) tri-valve system (e.g. Discofix^® C, B. Braun, Switzerland) that facilitates the change-over between both solutions; and (4) cannulation of the portal vein; B: Roller pump-mediated perfusion fixation at a flow of 1 mL/g liver tissue: (1) pre-perfusion buffer; (2) fixative solution; (3) tri-valve system; (4) low-flow peristaltic pump; and (5) cannulation of the portal vein; C: Injection or puncture perfusion fixation, using a needle to inject fixative into a wedge biopsy of about 1 cm × 1 cm × 1 cm dimensions. Injection to approach a rate of 1 mL/min: (1) Petri dish filled with 37°C physiological saline solution; (2) liver tissue wedge fixed by the tip of a forceps; and (3) syringe containing fixative solution; D: Different types of needles and their variants commonly used for portal vein insertion: (1) 18-23 G needle typically used for gravity-mediated perfusion for rat and mouse livers; (2) 18 G needle catheters are recommended for peristaltic-pump-mediated perfusion; (3) 12-13 G needles are typically used for larger animals such as rabbits; and (4) 25 G syringes are ideal for the injection of fixative solution in hepatic tissue. In case of accidental failure of procedure A or B, method C (injection perfusion) can be used to save the preparation.