Double Embedding

The term double embedding could refer to any processing system which uses two support media at the same time. Usually, however, it refers to Peterfyi's celloidin-paraffin wax technique. Gelatin-paraffin wax and agar-paraffin wax methods are usually referred to by those names, rather than the general term. It is advisable to check what form of double embedding was used if trying to follow the details of a project in which some variant of it has been employed.

The basis for these techniques is to obtain increased support to the tissues, the first inclusion procedure often being used to hold the tissues in place while the second infiltrates. It is not as cut and dried as that would infer, however.

Peterfyi's double embedding
In Peterfyi's technique, the celloidin is more than just a glue to stick the tissue pieces together. It also supports it in the sense of protecting it from the harsh effects of hot paraffin. The double embedding sequence results in less shrinkage than paraffin alone, but not as little as celloidin alone. It is, however, considerably faster.

1. Prepare a 1% solution of celloidin in methyl benzoate. This should have the consistency of honey.
2. Thoroughly fix the tissue and trim to an appropriate size no more than 3 mm thick.
3. Thoroughly dehydrate the tissue to the absolute ethanol stage.
4. Place in an equal parts methyl benzoate-ethanol mixture for a few hours.
5. Infiltrate with celloidin in methyl benzoate overnight.
6. Drain excess celloidin from the tissue and place tissue into xylene, 3 changes, an hour in each.
7. Infiltrate with paraffin wax at 60°C for 3 changes of 2 hours each with intermittent agitation.
8. Cast the block as for the paraffin infiltration technique.
9. Section, float out, stain and coverslip as for a paraffin block.

Kultchitzky's double embedding
Kultchitzky also proposed a technique, which involved regular celloidin processing, hardening with chloroform vapour, then infiltrating with paraffin wax. It is noted as being recommended for embryos and hard insects (flees, ticks).

1. Impregnate the tissue with celloidin according to a standard schedule, but harden the celloidin by exposing the block to chloroform vapour.
2. Trim the celloidin block and place it into chloroform or xylene.
3. Add some lower melting point paraffin wax to the block and clearing agent and leave to infiltrate at elevated temperature until properly infiltrated..
4. Pour off the clearing agent and wax mixture and replace with molten paraffin wax for a few hours until completely infiltrated. Repeat if necessary..
5. Cast the block as for the paraffin infiltration technique.
6. Trim the block so as to leave a rim of paraffin surrounding the celloidin. This facilitates ribboning.
7. Section, float out, stain and coverslip as for a paraffin block.

 

References
Gatenby, J. B. and Beams, H. W., (1950)
The Microtomist's Vade-Mecum Ed. 11, pp. 100-108, (para. 187-203).
J. & A Churchill Ltd., London, UK.

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