Fluka Durcupan, the new embedding agent, opens new roads in the observation of enzymatic digestion processes and for carrying out histochemical investigations in the submicroscopic range. It enables formalin and other aqueous fixatives to be used for electron microscopy and the dehydration of the tissue in alcohol or acetone to be dispensed with, so that excessive changes in the ultrastructure of the cells are obviated.
Fluka Durcupan is supplied in original packages, each package containing enough material for 10 embedding operations. The material comprises the following four components:
|100g of embedding substance, a water-soluble aliphatic polyepoxide (the former Experimental Product X 133/2097 of the CIBA Aktiengesellschaft, Base, Switzerland)|
|100g of 964 hardener, an anhydride of a diazide with aliphatic side chain|
|20g of 960 hardener, a phenol derivative with amino groups|
|20g of plasticizer (dibutyl phthalate)|
These four components make up a polymerizable mixture.
1. Fixing and dehydrating
Fix the tissue in, say, osmium tetroxide, potassium permanganate, or 10% formaldehyde (with Veronal buffer, pH 7 - 7.5).
Dehydrate it not in alcohol or acetone, but in a series of mixtures of water with Component A, the concentration of Component A increasing in consecutive stages, as shown in the following table:
|1st Tray||50% Component A with 50% water*||for 30 minutes||shake tray if possible|
|2nd Tray||70% Component A with 30% water*||for 45 minutes**||shake tray if possible|
|3rd Tray||90% Component A with 10% water*||for 45 minutes**||shake tray if possible|
|4th Tray||100% Component A||for 90 minutes**||shake tray if possible|
|5th Tray||100% Component A||for 90 minutes**||shake tray if possible|
* Aqueous solutions of the following contrasting agents may be added to the contents of these trays:
Osmium tetroxide, phosphotungstic acid, uranyl acetate. Do not add lead hydroxide (see section on Contrasting).
** These times may be varied without danger (e.g. up to 90 minutes in 2nd tray).
The dehydrated tissue is now placed in a polymerization mixture of the following composition:
Miscibility with Water
|5 ml|| |
|11.7 ml|| |
|1.0 - 1.2 ml (very critical!)***|| |
|0.2 - 0.4 ml|| |
*** Insufficient Component C: The block becomes too soft.
Excess Component C: The block becomes granular
Leave the tissue overnight at 4 - 5C in this comparatively viscous mixture. Its is advisable to shake it while in the mixture.
Now fill the gelatine capsules with the freshly prepared polymerization mixture of the above composition and charge them with the tissue specimens. Polymerization takes place at 37 - 45C and lasts 3-4 days.
The tissue fixed with formaldehyde shows a very low contrast in the electron microscope, an advantage for certain histochemical investigations. However, for purely morphological investigations, contrasting agents must be used. The following heavy-metal compounds are suitable for this purpose: osmium tetroxide, phosphotungstic acid, lead hydroxide and uranyl acetate. Contrasting can be carried out in two ways: Either let the mount mesh covered with ultrathin specimens float for about an hour on solutions of these heavy-metal compounds, or add the aqueous heavy-metal compound solution (but not lead hydroxide) to the dehydration bath trays marked *.
The blocks obtained according to this embedding method with Fluka Durcupan are mostly softer than methacrylate blocks and present certain difficulties in the preparation of ultrathin sections, particularly because of the chatter, a frequent occurrence. Ordinary glass knives or knives made of the harder Tempax glass are suitable for ultramicrotomy. (Diamond knives have not produced any better results than glass knives.) The sections can be caught in a solution of 20 - 50% acetone in water.
If the Durcupan blocks are too soft, try to cut the tissue out and rebed it in prepolymerized methacrylate. Such Durcupan / methacrylate blocks are usually much easier to cut.
Animal tissue can be embedded in Fluka Durcupan more easily than plant tissue.
Take great care when working with Durcupan, as this substance may cause skin irritation and allergic reactions. Work always with rubber gloves. Immediately wash off droplets on the skin with 3% boric acid solution. Wash hands, arms, face frequently in lukewarm soap water.