In the same way that you should ‘Think Before You Fix’, the choice of embedding media should be dictated by your required end-point. The basic principle is that by processing tissue into an embedding medium you harden the tissue and provide support protecting it from the mechanical forces associated with sectioning.
Parma ham and steak
A simple (and tasty!) comparison is to think about cutting thin slices from a fresh steak and compare that to cutting a thin slice from parma ham. It is of course much easier to cut thin slices from parma ham as the tissue (muscle mainly) has been processed by air-drying to preserve and harden the tissue. Preparing tissue for sectioning is a similar process except, that to produce micron rather than millimetre thin sections, we usually need to also provide a supporting medium such as paraffin wax or plastic.
Formalin Fixed Paraffin Embedded (FFPE) and IHC-P are synonymous with a large number of publications and product descriptions associated with tissue probing techniques such as immunohistochemistry and in-situ hybridisation. FFPE samples are an extremely versatile means for looking at the morphological appearance of cells within tissue sections. FFPE provides a means of producing thin sections (usually down to 2 µm in skilled hands) which provides good morphological and chemical preservation.
The rise of the antibody
FFPE also offers a good compromise for obtaining important morphological information on the spatial distribution of cells and tissue components. Most importantly, such a technique provides good preservation of protein, DNA and RNA consequently lending itself to a wide range of molecular tissue probing techniques. The last decade alone has seen a dramatic rise in the number of antibodies available for diagnostic and research applications and to identify biomarkers. Such an increase has only reinforced the popularity of FFPE and it is hard to imagine it being superseded.
The harder they are, the thinner they cut!
The harder the supporting medium, the thinner the sections can be cut. Thinner sections are generally associated with improved morphology (but reduced chemical reactivity). Epoxy resins offer high degrees of morphological preservation but limited opportunities for tissue probing and staining. However, the properties of epoxy resin can be controlled using different additives (such as dibutylphthalate) to soften the resin making it easier to section and produce semi-thin sections (~0.5-1 µm) for light microscopy. To produce the ultra thin sections for electron microscopic examination a harder formulation would be used. Various resin formulations are available that lend themselves to some forms of tissue probing and staining, but none have the versatility and ease of use of FFPE. Hard resins such as methacrylate can be used to embed processed bone samples and can be sectioned without decalcification on heavy duty microtomes.
The main advantage of cryostat sections is that the tissue is in a near natural state and may be suitable for the demonstration of sensitive antigens, although with a somewhat compromised morphology. Both these properties lend themselves well to the demonstration of fats and lipids which would normally be extracted during the processing into paraffin or certain resins and plastics, but again the principle holds in that the tissue is hardened (by freezing and cryopreservation in sucrose) to allow thin sections to be produced. The main difference here is that the sections thaw on contact with the microtome knife and are handled free floating, the big advantage is that much thicker sections can be made with good chemical preservation. It is particularly useful for looking at nerves in brain sections. The disadvantage is that manually handling free floating sections can be cumbersome compared to handling slide mounted sections.
What is that blob?
Freezing microtome sections could be considered to be at the opposite end of the spectrum from resin, in that the tissue is still pretty soft, enabling thick sections to be produced, but the softness means that the section normally can’t support itself as a section, instead it concertinas up into a unrecognisable blob that only when lifted into a water bath can it float out and reveal its true shape. Because of the minimal processing involved, these sections can be very sensitive for looking at the detection of proteins by immunohistochemistry or immunfluorescence.
|Paraffin||Versatile. Good preservation of morphology, proteins, DNA and RNA.||Not much! Can’t really produce sections thinner than 2 µm.|
|Epoxy resin||Good preservation of morphology. Can produce ultra-thin sections.||Limited for tissue probing and staining.|
|Cryo||Near natural state of tissue. Very sensitive for protein detection by IHC/IF.||Somewhat compromised morphology. Difficult to handle free-floating sections.|
Although not a definitive rule, hard embedding media are associated with good preservation and lower chemical activity whereas soft embedding media are the opposite. Paraffin wax is quite hard (but not too hard) and certainly allows for the demonstration of many cell and tissue constituents using molecular probing techniques or empirical staining methods.
When not embedding tissue samples in paraffin wax, we like to add some other extras- a wick, some scent, a source of ignition, some wine, chocolates, a hot bath, subdued lighting, a friend (If you’re lucky!) and…….relax after a hard day in the lab…..what is there not to like about paraffin wax!
Would you like to learn more about any of these techniques? Just let us know.