Paraffin processing and embedding

Paraffin processing is the common route to preparing slides for histological analysis. It is perfect for haematoxylin and eosin (H&E) staining as well as most special stains. Paraffin processing provides excellent resolution of cellular structures, if tissues are handled correctly at the fixation stage. Processing allows paraffin to infiltrate down to the cellular level, which gives the tissue enough even rigidity to be thinly sectioned and placed on a slide for further analysis.

Please adequately fix your samples and transfer them to 70% ethanol prior to bring them to the core. They can stay in 70% ethanol for an extended period of time without harming the sample.

Fixed tissue is first dehydrated through graded alcohols (70, 90, 95%), this is because wax and water do not mix. The tissues are then placed in xylene, which acts as a linking agent between the alcohols and wax. Finally the tissues are impregnated with paraffin wax at a temperature just above the melting point of the wax being used, and under vacuum. How much time and whether vacuum or heat are used, at each stage, is dependent on the type of tissues being processed. Human tissue often requires much longer in each stage plus the additional vacuum and heat however, if you use the vacuum and pressure on mouse tissues they often become very dry and difficult to section.

Tissues are then embedded in molten paraffin wax. This is a vital stage of tissue preparation, the positioning of the tissue in the mold is very important to getting what you want to see on the slide. Homogenous organs, like liver and spleen, are embedded on the flattest surface, more heterogenous tissues, like skin, hearts and brain can be placed in multiple orientations to ultimately section onto a slide the exact area of interest

Commonly special embedded samples:
- Embryo sectioning for 4-chamber heart view are embedded on their heads (transversely)
- Skin - embedded 'on edge' to see through the epidermis, dermis and subcutous
- Hearts - if 4-chamber view is required hearts are embedded with the front of the heart on the cutting surface with a slight tilt towards the atria
- Kidneys - Bisected through the renal pelvis and embedded flat surface down, resulting section will show both corex and medulla
- Brain - if a coronal view of the hippocampus is required the brain is sliced at the olfactory bulbs (at the front) that is then embedded flat surface down.

Once the wax has cooled the resulting tissue 'blocks' can be sectioned on the microtome. Please see tissue sectioning for more details.

There is a degree of shrinkage in the tissues due to the dehydration, this should be taken into account when designing experiments that require absolute measurements.
Common exceptions to paraffin processing:
- Oil Red O staining for lipids. The processing solutions remove the lipids from tissues rendering the special stain ineffective.
- X-Gal staining. Formalin fixation kills the functioning enzyme required to complete this technique
- Some antibody staining will not work on paraffin processed tissues, which ones are determined empirically.  A commercially purchased antibodies data sheet should give you some indication of whether it will work on paraffin, frozen or plated cells, but in some cases their information may not be true when tried in your own lab. Antigen retrieval may be performed to unmask epitopes on paraffin tissue
- Immunofluorescence is often, but not always performed on frozen tissue due to the high background auto-fluorscence seen in formalin fixed paraffin embedded tissue
please see frozen processing for these techniques
Please contact the core with more questions

Resources
- Stains file, an all together good resource for histological technique: http://stainsfile.info/StainsFile/prepare/process/processing.htm
Dako processing chapter (written by John Kiernan): http://www.dako.com/08066_12may10_webchapter16.pdf