Embryo collection - Epoxy resin TAAB PREMIX

Embryos were fixed, washed, treated and embedded in epoxy resin

Protocol

Fixation

Embryos were fixed in 2.5% Glutaraldehyde in 0.1M Cacodylate Buffer pH 7.3 + 0.1M Sucrose. 2Hrs for 24 hours.

Wash

Embryos were washed in 0.1M Cacodylate Buffer + 0.1M Sucrose for 2x10 mins. Embryos for storage were kept in this solution at 4°C.

Dehydration

Increasing concentrations of ethanol in distilled water were used to dehydrate the embryos. 10%,30%,50%,70%90% ethanol each for 2x10mins 0-5day and 2x30mins for 6-8 day before a final 3 changes of 100% dry ethanol.

Infiltration

Due to the very fragile nature of these young embryos, Propylene Oxide was omitted as a transition fluid for the epoxy resin. Instead mixtures of TAAB PREMIX plus accelerator in 'dry' ethanol (1:2, 1:1 and 2:1 for 2 hrs each) were used to assist the infiltration.

All embryos were then transferred into a thin layer of complete resin overnight at room temperature to infiltrate.

At the same time a thin layer of TAAB PREMIX plus accelerator was poured onto the bottom of the final embedding moulds (flat aluminium foil dishes) and polymerised overnight at 60°C to give the bottom layer of the 'sandwich' technique used for embedding.

Embedding

After the infiltration stage was complete the embryos were transferred to fresh TAAB PREMIX plus accelerator that had been added to the foil dishes as another thin layer. The embryos were allowed to sink to the interface of the two layers, carefully orientated with a fine glass rod, then placed in a 60°C oven for 3 days to polymerise.

Sectioning

The embryos were then subject to histology sectioning