Fixative: 2.5% glutaraldehyde, 1.5% paraformaldehyde, 0.5mM CaC12 in caco 0.IM or PBS O.IM buffer, pH 7.2-7.4. Addition of fixative directly on the cells in suspension and horizontal agitation. (24 hours at room temperature). Renew the fixer before nightfall. The next morning, renew the fixative and continue fixing with horizontal agitation at room temperature for another 24 hours. Total time of this fixation: 48 hours.

Remove the supernatant, add 1 or 2 drops of 2% agarose, resuspend and centrifuge while hot, 3-4 min.

Cool on ice.

Cut the pellet into tiny pieces and place them in the O.1M caco buffer.

Wash in caco 0.1 M, 2×20 min. (minimum) at room temperature. They can also be left there for the night or the weekend at 4°C.

Post-fixation: Os04 1% in caco O.1M, 90 min., at room temperature.

Wash with cocoa O.1M, 3×10 min., at room temperature, or at night or on weekends, at 4°C.

Dehydration: 30% alcohol, 50% alcohol, 70% alcohol, 95% alcohol, 100% alcohol, 10 min. each. 100% alcohol, 40 min., 100% alcohol 10 min.

Perform 2 passages of 15 min each in propylene oxide.

Mixture of propylene oxide and epon (3:1) without DMP, at night room temperature.

Mixture of propylene oxide and epon (1:1) without DMP, at night room temperature.

Mixture of propylene oxide and epon (1:3) without DMP, at night room temperature.

Transfer to pure epon WITHOUT DMP for the day.

Transfer to pure epon WITH DMP for about 5 hours.

Coat.