FIXATIVES

Preparation of 4% Formaldehyde in 0.1 M Phosphate buffer:

• 4 g formaldehyde powder in 45 ml water
• Under stirring, heat to 60°C
• Add 3-5 drops of NaOH 1N, until solution becomes transparent
• Add 50ml 0.2 M Phosphate buffer
• If mixture is at room temperature, adjust pH to 7.4
• Adjust volume to 100 ml
   

Standard perfusion fixation (under 20 cm water pressure)

• Rinse 5-10 sec with PBS-Ca²⁺
• Fix with 1.5 % glutaraldehyde in 0.1 M Phosphate buffer for 10 min; pH 7.4
• Remove liver and cut well perfused lobes, immerge for 1h in 4 % formaldehyde at 4°C
• Cut small pieces (1 x 1 x 1 mm) in fixator
   

Perfusion fixation for immunolocalization (under 20 cm water pressure)

• Rinse 5-10 sec with PBS-Ca²⁺
• Fix with 4 % formaldehyde and 0.1 % glutaraldehyde in 0.1 M Phosphate buffer for 10 min; pH 7.4
• Remove liver and cut well perfused lobes, immerge for 1h in 4 % formaldehyde at 4°C
• Cut small pieces (1 x 1 x 1 mm) in fixator
   

Cell cultures:

• Rinse 3 - 5 times with PBS-Ca²⁺ at 37°C
• Fix with 1.5 % glutaraldehyde in 0.1 M Phosphate buffer for 20 min, 22°C, pH 7.4

OR FOR IMMUNOLABELING

• Fix with 4 % formaldehyde and 0.1 % glutaraldehyde in 0.1 M Phosphate buffer for 20 min, 22°C, pH 7.4