In the fields of medical science, such as anatomy
, tissue sections are often embedded in paraffin wax then frozen before their use in studies in order to store them for some time. Wax holds the tissue together so that a microtome can slice it into sections thin enough for light to pass through for microscopy analysis. Typically, the paraffin
-embedded tissues are sectioned into +/- 5 micron
s thick then mounted onto slides by heating in a laboratory
oven, where the wax slightly melts. In order for any treatments or tests to work on the tissue
sections, they must be completely deparaffinized. A sample deparaffinization
outline is thus:
Each step involves the soaking of the slide-mounted sections in the specified solution.
1. Deparaffinize in Xylene, 3 changes x 5 minutes.
2. Hydrate in 100% Ethanol, 2 changes x 2 minutes.
3. Hydrate in 95% Ethanol, 2 change x 2 minutes.
4. Hydrate in 70% Ethanol, 1 change x 1 - 2 minutes.
5. Hydrate in 50% Ethanol, 1 change x 15 minutes.
6. Rinse in running tap water, 1 x 10 minutes.
7. Pretreat sections in sodium-citrate buffer to unmask antigenic determinants masked by the paraffin. (5.98g Sodium Citrate/2L distilled water adjusted to pH 6.0 using HCl)
8. Submerge slides in citrate buffer and bring to a boil. Keep the slides at a temperature between 90 and 95 degrees C.
9. Place slides in a rack and submerge in the citrate buffer for 30 minutes, carefully watching that the sections do not come off the slides.
10. Remove slides and wash in PBS buffer solution (pH 7.5) 1 x 5 minutes.