Paraffin sectioning is the most frequently used method for histological section preparation. However, there are some questions that we often encounter while cutting the paraffin-embedded tissue.Why can’t wax block be trimmed smoothly? Why section breakage and coiling are easy to occur during the cutting? Why the sections can’t be flattened during spreading? This text will provide you with the detailed methods of cutting qualified paraffin sections.
After tissue separation, it is required to fix it within 1 hour.
The stationary liquid shall be 4 to 10 times the volume of the tissue and have an accurate concentration. Excessively thin and rich liquid will affect tissue form and fixation effect. In case of discovering any stationary liquid deterioration, such as white precipitation of formaldehyde liquid, it is required to conduct immediate replacement.
The fixation temperature should be the ambient temperature or in a fully enclosed tissue processor of 35-37°C. Excessively high temperature will accelerate tissue autolysis and excessive contraction and destroy the antigen and DNA in the cells.
The fixation time shall not exceed 40 hours and shall be adjusted according to the ambient temperature and specimens.
The key to dehydration is make the low-concentration alcohol dehydration time match the high-concentration alcohol dehydration time. Low-concentration alcohol can reduce tissue shrinkage and make it easier to cut. High-concentration alcohol can dehydrate tissues even more.
Generally, the specimen dehydration time (35°C) is 1.5 hours for 75% alcohol, 2 hours for 85% alcohol, 1.5 to 2 hours for 95% alcohol (twice), and 1.5 to 2 hours for pure alcohol (twice). The dehydration time of small specimens is little different from that of large specimens. Normally, there’s no need to separate them (often, brittle small specimen is a result of thick paper package or sticking with each other, which leads to incomplete tissue dehydration. If the time is urgent, it’s allowed to properly shorten the dehydration time. The duration of dehydration is closely related to the ambient temperature, the thickness and type of the tissue, the degree of tissue fixation, etc.).
The thoroughness of dehydration is directly associated with tissue transparency. If the temperature is too high (beyond 50°C) or acetone is used during dehydration, it can easily lead to excessive tissue shrinkage and hardened and brittle tissues. There are three reasons for brittle tissue caused by tissue dehydration:
False fragility: due to insufficient dehydration, the paraffin cannot penetrate into the tissues when the tissues are immersed in wax. If the tissues are “baked” under high temperature, it will become hard and brittle. The sections will have powdery or filamentary parts. Hard tissues such as uterine leiomyoma will also have edges, and the whole section will collapse outward in a worse case. The tissues with seriously insufficient dehydration become soft and even have water.
Operation Points of Paraffin Sectioning
Before sectioning, tighten the relevant locking rod or screw on the microtome, otherwise, the section skipping, and uneven thickness of sections may occur.
Scrape the remaining paraffin around the embedding box, otherwise the sample clamping may be loose, affecting the quality of the section.
Correctly trim the block and carry out rough trimming first. The thickness is about 15-30 microns, and the harder tissue or smaller tissue should be thinner. After rough trimming until all tissues are exposed, fine trimming can be carried out.
Paraffin block freezing mode is selected, and the ice box is superior to the freezing table. Use an ice box to wet paraffin blocks by adding water. It has the advantages of fast freezing speed, small size, low use cost, and no external power supply.
During paraffin sectioning, gently hold the hand wheel with even and gentle force, and the shaking should not be too quick or too slow. If the speed is too high, it will lead to uneven section thickness and has difficulty expanding the section. If the speed is too low, it will thicken the section. The sections shall be complete, thin and even.
After paraffin sectioning, put the section into cold water first, and then move it to hot water, so that the section will be thinner, with fewer wrinkles and bubbles. The water temperature of the spread slice shall be between 42-55°C, which is related to the melting point of the embedding paraffin. If the water temperature is too high, the tissues and cells will disperse. If the water temperature is too low, the wrinkles of the sections cannot be flattened.