2.1.2. Frozen section fixation

Frozen section is the rapid way to fix and mount histological sections. Fresh or fixed tissues are snap-frozen in liquid nitrogen, or with a high pressure jet of CO₂, and sectioned using a refrigeration device called a cryostat. The frozen tissue is sliced and the frozen slices are mounted on a glass slide and stained. The tissues are infiltrated in 40% sucrose prior to freezing and sectioning to help protect the tissue during freezing.

Advantages of fixation by frozen sections

• •  Give better preservation of antigenicity

•  Minimal exposure to fixative

•  Not exposed to the organic solvents

•  Disadvantages of fixation by frozen sections

•  Lack morphological detail

•  Possibility of biohazard

 

2.2. Dehydration

Dehydration removes fixative and water from the tissue and replaces them with dehydrating fluid. There are a variety of compounds used, many of which are alcohols. To minimize tissue distortion from diffusion currents, delicate specimens are dehydrated in a graded ethanol series from water through 10%-20%-50%-95%-100% ethanol.

In the paraffin wax method, following any necessary post fixation treatment, dehydration from aqueous fixatives is usually initiated in 60% -70% ethanol, progressing through 90%-95% ethanol, and then two or three changes of absolute ethanol before proceeding to the clearing stage. Examples of dehydrating agents used are ethanol, methanol, and acetone.

The duration of dehydration should be kept to the minimum consistent with the tissues being processed. Tissue blocks 1 mm thick should receive up to 30 minutes in each alcohol, blocks 5 mm thick require up to 90 minutes or longer in each change. Tissues may be held and stored indefinitely in 70% ethanol without harm.