Fixation methods for electron microscopy of human and other liver

Table 2 Addendum summarizing chemicals necessary for fixation of liver tissue

Compound	Quantity
Cacodylate buffer 0.2 mol/L, pH 7.4	700 mL
Sodium cacodylate trihydrate (Sigma, C0250)	22.9 g
MQ-(purified by ion-exchange)-water	674.8 mL
HCl (0.2 mol/L)	25.2 mL
Filter through 0.2 μm Millipore or Wattman filter paper
Cacodylate buffer 0.15 mol/L, pH 7.4
Cacodylate buffer 0.2 mol/L	900 mL
MQ-water	300 mL
Filter through 0.2 μm Millipore or Wattman filter paper
Glutaraldehyde fixative, 1.5% in cacodylate buffer 0.067 mol/L
Cacodylate buffer 0.2 mol/L	575 mL
Sucrose (Sigma, S0389)	17.215 g
Glutaraldehyde solution 70% (Sigma, G7776)	36.96 mL
MQ-water	1113.04 mL
Filter
Osmium tetroxide 1% in phosphate buffer1	90 mL (6 × 15 mL)
Osmium tetroxide 4% for EM (Sigma, 75632)	22.5 mL
Phosphate buffer 0.2 mol/L	45 mL
MQ-water	22.5 mL
Washing buffer 1
Cacodylate buffer 0.2 mol/L	30 mL
Sucrose	0.9 g
MQ-water	60 mL
Washing buffer 2
Phosphate buffer 0.2 mol/L	45 mL
MQ-water	45 mL
Phosphate buffer 0.2 mol/L pH 7.4 (200 mL)	200 mL
0.2 mol/L Na2HPO4.2H2O (17.8 g in 500 mL MQ-water)	162 mL
0.2 mol/L NaH2PO4.H2O (13.8 g in 500 mL MQ-water)	38 mL

Fluids and chemicals (volumes to be adapted to needs).

¹An alternative to the osmium postfixation is addition of 1.5% potassium ferrocyanide to 1% osmium in veronal acetate buffer (pH 7.4) at 4°C for 2 h, which strongly enhances the contrast of membranes in cells and tissues[20] (see Figure 13).