Paraffin Embedding Protocol

Scientists usually need to solidify biological samples to section them off. This makes it easier for scientists to add dyes, antibodies and probes to the biological sample. It also reduces the overlay of various cell layers. Scientists use paraffin wax as a hard matrix for the cutting, since the paraffin does not mix with water. Paraffin is a type of hydrocarbon. The biological samples contain water, so the scientists must dehydrate the samples using increasingly more concentrated ethanol baths.

Xylene

• Scientists remove the ethanol using xylene. Then they use molten paraffin wax to remove the xylene. For light microscopy, the paraffin sections are usually five μm thick. Paraffin wax isn't hard enough for thinner slices. Scientists rehydrate the tissue by using xylene first, then ethanol. Then they rinse the biological sample with distilled water to avoid contamination.

Fixing

• Paraffin-embedded tissues are usually fixed using neutral buffered formalin, which is the commercial version of formaldehyde. This includes paraformaldehyde and methanol, which prevents the formaldehyde from converting into formic acid. Thinly sliced tissues need 48 hours of fixation at room temperature for optimal histology, which is the study of microscopic anatomy. Otherwise, the tissue will harden and become brittle. Before processing, the tissue is usually stored in 70 percent ethanol at a temperature of 4 degrees Celsius.

Paraffin Processors

• Scientists often use paraffin processors to send the tissue through baths that gradually dehydrate the material before hot paraffin can enter through the tissues. Scientists run the processor overnight. The processor only heats the tissue for a certain length of time dependent on the tissue to make it doesn't become hard and brittle. The processor uses a vacuum that speeds up the permeation of paraffin and removes air bubbles. Once processed, the tissues are stored in cassettes at room temperature.

Wax Melting

• The paraffin melts for one hour prior to adding the tissue. The whole cassette is placed in a 65 degree Celsius paraffin bath for 15 minutes. A cold aluminum heat sink cools the paraffin wax for 20 minutes. When the wax cracks or the tissues aren't aligned correctly, the scientist melts the paraffin and repeats the process.

Cutting

• When sectioning the tissues, the scientist gets the water bath to 35 to 37 degrees Celsius. The water is fresh and deionized. The blocks are placed face down on an ice block or heat sink for 10 minutes. A blade cuts the block into sections. Two problems that can occur are poor ribboning and fragmenting specimens. When the specimens fragment, the water is too hot. Whether the block does not ribbon well, you should put it back on the ice block and firm up the wax before starting again.