Fixation: Specimens may be routinely fixed in 10% neutral
buffered formalin (NBF). Fixation should be commenced as soon after
surgical excision as is practically possible to prevent autolysis and bacterial
decomposition.
Formalin fixation should ideally be allowed to proceed for at least 12
hours before processing. It is largely reversible by water washing
until the specimens have been in formalin for more than 24-28 hours. Specimens
may be stored in NBF.
It is important to use NBF routinely as it stops the formation of formalin
pigment in the tissue (caused by acidity in the fixative solution).
This would otherwise present as artefact in the final stained sections.
Very small specimens may alternatively be fixed in Bouin's
fluid. Picric acid in the Bouin's fluid colours the tissue yellow
temporarily, this facilitates subsequent handling before and after processing,
and is easily removed from cut sections by alcohol in the initial steps
of the staining procedure. It should be noted that tissues fixed
in Bouin's tend to stain more brightly than those fixed in NBF.
Decalcification: After fixation bone and other calcified tissues
are treated with 10% formic acid to remove the calcium
content. Tissues may be left standing in the acid for several
weeks, changing the acid weekly until a chemical test for dissolved calcium
is negative. Large specimens can then be further cut and trimmed
to expose appropriate structures for subsequent processing.
Phenol Solution: Other hard or dense material
such as tendon, nail, decalcified bony structures etc., may benefit from
pre-treatment with a mixture of 4% phenol in 70% alcohol (ie: 4g phenol
dissolved into 100ml 70% alcohol), before final processing. This
helps to soften the tissues for easier sectioning. It can also be
used to 'rescue' tissue that has been accidentally dried out. The
length of time the specimen is left in the solution depends on how badly
dehydrated or hard the tissue is but generally 12-18 hours will be sufficient.
Some softening of tissue and further surface decalcification may be performed
on the wax embedded tissue if required. (Trimmed block surfaces are treated
with a commercial solution of softening fluids known as Mollifex®
immediately before cutting.)
University
of Bristol
