Processing tissues for histology. University of Bristol, Department of Clinical Veterinary Science.

University of Bristol

PROCESSING SCHEDULE FOR BIOPSY SPECIMENS

Overnight 18 hour schedule, from 3 pm to 9 am, using an automatic tissue processor (Shandon Citadel^®, Leica Histokinette^®etc).

The tissues are placed in labelled plastic cassettes marked using a lead pencil which survives the processing fluid/solvents or in larger cassettes with labelled cards. The processing machine automatically transfers the cassettes, in a basket through the following:

Steps 1-6 give gentle, but complete dehydration to remove aqueous fixative and any tissue water content.

Steps 7-9 is the 'clearing' fluid which is totally miscible with the dehydrating alcohol and wax embedding agent. Other clearing fluids are xylene and chloroform but Histo-Clear^® is safer to use routinely. This solvent may also be referred to as a 'link reagent'.

Steps 10-11 impregnate the tissue with molten wax for the final embedding stage which sets specimens in blocks of paraffin wax from which sections may be cut.

Embedding, the final stage of the process, is carried out on a Tissue Tek^® embedding centre using stainless steel cassette moulds of several routine sizes (suitable for large numbers of small and moderate dimensions). The plastic labelled cassettes are incorporated into the finished block which makes it more rigid for sectioning. It also ensures that the specimen always stays with its proper given number. The blocks are finally cooled on an integral cold plate and removed from the mould once the wax is set hard.

The tissues, trimmed prior to processing to fit inside cassettes (25 x 20 x 4 mm maximum), may have shrunk considerably after the process and are orientated in the wax mould to present required features/planes on the final cutting surface.

Sections are cut from the blocks using a microtome which may be a rotary or sledge type .

Sections are generally cut 4-5 microns but may be thicker for certain tissues such as CNS.

Cut sections are floated onto a warm water bath to smooth out the creases and then lifted out onto a glass microscope slide and allowed to dry on a hot plate set just below the melting point of the wax .

Once the sections are dried onto the slide (about 1 hour for most tissues) they can be stained. All tissues are routinely stained with the haematoxylin and eosin method. A large number of other staining techniques are available to identify many different tissues components.

Stained slides are mounted under a glass coverslip using a synthetic mounting medium such as DPX.