Tissue embedding in resin, Technovit 7100 - The world under the microscope

Tissue embedding in plastic, Technovit 7100

Paraffin embedding is common in most histological laboratories. However, in certain cases it is desirable to embed tissue in plastic. In this way, a much thinner coupe can be cut with a plastic technique and a much better cell morphology is achieved. Plastics for histology come in three forms: GMA (glycol methacrylate), MMA (methyl methacrylate) and epoxy. The GMA, such as Technovit, is a relatively soft type of plastic with which a section thickness between 0.5-1µm can be achieved. These sections are also referred to as the semi-thin sections. GMA can be cut on a rotary or ultramicrotome with steel, glass or diamond blades. Of the steel knives only the 'D' profile knives can be used.

The MMA plastic, such as Araldit and LR White, is harder and can therefore be cut even thinner (50-70nm). The MMA plastics are only cut with glass or diamond knives on an ultramicrotome. An epoxy is e.g. Epon. Epon is hard and tissue which is secondary fixed with osmium tetroxy can be embedded without any problems. These coupes are made for electron microscopy.

Some advantages and disadvantages of paraffin compared to plastic are shown in the table below,

Paraffin	Plastic
standard in histology	is used less frequently
Quite fast machining time and easy to use	takes a lot of time and very precise work has to be done
large sections possible	only small coupes possible
many different stains can be used	few stains to use
can be cut with cheap disposable knives	knives are expensive
coupe thickness limited to 2-3µm	coupe thickness accessible between 50nm and 1µm
due to the large shrinkage usually many artifacts in section	little shrinkage, very good morphology
can be cut on a simple rotary or sledge microtome	GMA should be cut at least on a rotary microtome with retraction, MMA should be cut on an ultramicrotome.
hard tissues are difficult to cut	hard tissues are easy to cut

The company Kulzer from Germany has a GMA plastic, Technovit 7100, in its program that is excellent to use for a amateur histologist. A Brochure of Kulzer from 2011 can be downloaded in 'Downloads'. This plastic can be ordered from e.g. Morphisto in Germany: https://www.morphisto.de/en/shop/detail/d/TECHNOVIT%C2%AE_7100//11823/.

The embedding protocol below is based on the embedding of tissue with Technovit 7100.

tissue blocks should not be larger than ≈ 6 x 10mm and ≈ 5mm high; tissue fixation in formaldehyde 4%; rinse fixed tissue in running tap water (24 hours); dehydrate tissue in ethanol series 50%, 70%, 85%, 95% and 100% (I use 12 or 24 hour steps); infiltrate with a solution of ethanol 100% and Technovit 7100, 1:1, 24 hours; 100ml Technovit mix (15minutes) with 1gram harder I ; infiltrate tissue into this solution for 48 hours (room temperature); take fresh infiltration solution and allow the tissue to infiltrate again for 48 hours; mix 15 parts of infiltration solution with 1 part harder II (polymer solution); place the tissue in the mold and fill with the polymer solution; allow to polymerize for 1 hour at room temperature; polymerize for 2 hours at 37º Celsius; place the plastic block holder upside down on the mold; mix 2 to 3 parts Technovit 3040 powder with 1 part Technovit standard (work quickly because this solution is already viscous after 1 minute and hard after 2 minutes); remove the blocks from the mold after 15 minutes and cut immediately.
	It is advisable to take consumables made of glass. This is to minimize contamination of the Technovit.
	Use of glass pasteur pipettes to apply Technovit with hardener II in the mold. To apply the Technovit 3040, it is useful to remove a pipette from the narrow outlet and use it in this way.
	15 parts infiltration-Technovit with 1 part hardener II can be mixed well in a small size test tube.
	An adjustable micro pipette of e.g. 200-1000µl is useful to measure e.g. 15 parts infiltration solution and 1 part hardener II.
	Technovit 7100, bags of 1gram harder I and 100ml harder II.
	The mold procedure with Technovit 3040 in view.
	Technovit 3040 powder and Technovit standard.
	Five different pieces of tissue are to be polymerized in Technovit.
	The Technovit 3040 is molded into the inverted block holders. This makes the polymerized technovit with woven stiffly connected to the block holders.
	After about 15 minutes, the blocks can be twisted out of the mold. I call twisting because the blocks can be quite stuck in the mold. Because the molds are slightly tapered, the removal is simplified.