[Histonet] Chitin Softening- Results

From:"Prior, A.E."

A few months ago I wrote asking for help regarding sectioning the eyes from Edible Crabs. Many thanks for all the suggestions. In between my proper work I managed to test a few of these out, and these are the results in brief:
 All eyes were collected fresh and fixed for at least 48 hours in formal saline (10%).Dehydrations were done using propan-2-ol rather than ethanol to reduce hardening effects. Embedded in Paramat wax via Histoclear.
 The shell of the crab is a mix of calcium and chitin. Eyes were placed in 10% formic acid (18-24 hours with 2 changes)under vacuum, until soft and all calium removed.
 Eyes were then treated with either sodium hydoxide, phenol/chloral hydrate mix or Nair ( a commercial hair removal cream available from all good chemists (that's a drug store to all you Americans))
 NaOH (24 h in 2% soln.)treatment softened the chitin enough so sections could be cut with extreme care. 
 The phenol treatment was less successful. Even after a week in the mix the chitin was still too hard to section properly. Tissue preservation was poor in these samples, but unsure whether this was solely due to the phenol.
 Coating the eyes in Nair for 24 hours produced eyes that section well. Slower speeds than normal were needed to avoid thickness variation in sections, but was quicker than the NaOH treated eyes. Tissue preservation was excellent as the Nair did not penetrate the eyes.

Thanks again for all the help on this. Hope these results are useful to people - I have the protocols if people need further details, contact me directly.

Andrew Prior
Biology Dept. 
Leicester University

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