Histology Techniques - Tissue Processing and Embedding

Tissue processing, is required next step after fixation to allow tissue to be embedded in molten wax. There are three main stages involved in tissue processing.  These are Fixation to make sure tissue is fixed properly in the required fixative, Dehydration via ethanol to remove any water in the tissue, Clearing to replace any water or ethanol with xylene, and finally Impregnation with molten wax, which are described on the chart below.

This procedure is required as tissue containing water is not normally miscible with paraffin wax. This is normally carried out on a processing machine in larger laboratories as it is more efficient and faster than by hand.

Paraffin wax does not provide a sufficiently hard matrix for cutting very thin sections for electron microscopy. Instead, resins are used. Epoxy resins are the most commonly employed embedding media, but acrylic resins are also used, particularly where immunohistochemistry is being used.

One benefit of using resin is that thinnner sections (0.35μm to 5μm) of resin-embedded tissue can also be cut for light microscopy.

One difference from the paraffin based technique is due to the immiscibility of most epoxy and acrylic resins with water this requires instead the use of dehydration, usually with ethanol.

Below are a list of machine schedules that can be used for processing.

Processing Schedules
Short fixation - Small Biopsy Use	Time In Reagent (Minutes)	Overnight fixation - medium tissue	Time In Reagent (Minutes)	3 day fixation - Supersize tissue	Time In Reagent (Minutes)
Formalin	15	Formalin	120	Formalin	60
Ethanol	5	Ethanol	50	Ethanol	480
Ethanol	5	Ethanol	50	Ethanol	300
Ethanol	10	Ethanol	50	Ethanol	300
Ethanol	10	Ethanol	50	Ethanol	300
Ethanol	10	Ethanol	50	Ethanol	300
Xylene	5	Ethanol	50	Xylene	300
Xylene	10	Xylene	50	Xylene	300
Xylene	10	Xylene	50	Xylene	300
Wax	10	Xylene	50	Wax	200
Wax	15	Wax	60	Wax	200
Wax	15	Wax	90	Wax	200
-	-	Wax	90	-	-

The processing schedules can be either hand processed or machine processed. It is normally easier to machine process as this can be done in a vacuum which speeds up the process, compared to hand processing. An alternate schedule that can be used for resin embedded tissue is listed below

Renal Resin Processing Schedule
Solution and Why	Time In Reagent (Minutes)
Formalin Used to fix the tissue as renal biopsies are often delivered fresh in saline.	45
70% Alcohol – Used to dehydrate the tissue as LR White is not miscible with water.	120
1:1 Absolute Alcohol: LRW – Used to dehydrate further the tissue, while the LR white is used to begin impregnation of the tissue	30
LR White – Used to further impregnate the tissue	45

 

After processing of tissue, it is then ready to be embedded into a wax block, which is the next step before it is ready for cutting. In major laboratories a large scale embedding centre is used for this work, as shown in the picture below.

On the embedding centre, the tissue is placed into a wax filled metallic mould and then placed on Ice to cool before it is formed as shown above. A Cassette is often used to provide a structure and support for the tissue for later microtomy.