Sections from 5 to 15 µm in thickness are suitable for light microscopy studies. To get them, the sample has to be hardened, but not get fragile. This can be done by freezing or by embedding the samples in some substances. Embedding means to infiltrate the sample with a liquid solution that gets solid without affecting, or altering minimally, the sample features.
The most common embedding compound in the histology labs is paraffin. It is a mix of hydrocarbons that has a candle wax-like consistency at room temperature. There are several types of paraffin with different fatty acid length, which determines the fusion temperature. The range of paraffin melting is from about 45 ºC to 60 ºC.
Procedure
Fixation. Bouin, formaldehyde and FAA: formaldehyde / acetic acid/ alcohol are generally used.
Trim or select the sample: the preferred size is about 0.5 cm.
Dehydration of the samples: ethanol.
Dehydration is necessary because paraffin is not miscible with water, and therefore the water of the sample has to be removed.
3x10 min in distilled H2O
4x10 min in ethanol 70º
4x10 min in ethanol 80º
4x10 min in ethanol 90º
4x10 min in ethanol 96º
5x10 min in ethanol 100º
Organic solvent: xylene
Paraffin is not miscible with ethanol. Xylene is miscible with both ethanol and paraffin. Thus, xylene is the intermediary between ethanol and paraffin.
4x5 min in xylene
Liquid paraffin (in stove at 60 ºC).
60 min paraffin I
60 min paraffin II
60 min paraffin III
Put the sample within a heated mold and add liquid paraffin to fill the mold. Leave them could down at room temperature to get paraffin solid.
Notes
Fixation: the sample needs to be fixed before the beginning of the embedding procedure. Many fixatives can be used, but the most common are formaldehyde, Bouin, Carnoy, Karnovsky and FAA. Fixatives containing glutaraldehyde are not the best for paraffin embedding.
Sample selection: samples larger than 1 cm are not recommended because the diffusion is slow and the inner part of the sample may not be properly embedded. All the steps of embedding, dehydration, xylene, and paraffin, are adapted to the sample size. The larger the sample, the longer the time.
Sample selection: if the sample is hard, some treatment has to be done to soften the tissues. For example, minerals need to be removed from bone tissue before dehydration begins.
Dehydration: if the fixative contains picric acid, it is important to remove it as much as possible during the first dehydration steps, which can be tested by the fading of the yellow color.
Dehydration: it is a good practice to perform the dehydration and xylene steps in gentle agitation. The movement of the sample in the liquids favors the diffusion processes between the inner part of the sample and the liquid.
Paraffin block: depending on the type of paraffin microtome, it is necessary to attach the paraffin block, containing the sample, to special supports that fit to the arm holding the paraffin block.
Products
Ethanol 70º, 80º, 90º, 96º y 100º
Xylene
Paraffin
Labware
Beaker
Forceps
Stove 60ºC
Paraffin mold
Embedding cassette