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I. Summary
Tissues need to be processed through solvents with decreasing water concentrations inorder to infiltrate
them with paraplast. You will transfer your tissues through increasing alcoholic solutions. They will then
be "cleared". The clearing agent is soluble in both alcohol and paraplast. The will then be infiltrated with
Paraplast, blocked and stored until sectioning.
Dehydration Methods:
Several different methods are available. Alcohol is the most common solvent used for both dehydration and
rehydration. Dioxane is also a clearing agent. It provides for a more rapid processing of tissue and functions
as a clearing agent, but is also toxic. See details below under clearing agents.
II. Protocol
We will use the following schedule to process our tissue. No Times are listed. Please fill in
Embedding at _____________________ PM
For Tissue preparation one to two hours in each solution should be adequate. Tissues with a high
water content such as embryo tissue would require a much shorter time. To ensure complete
removal of water during dehydration, use two changes of 100% ethanol of at least one half hour
each. Never leave tissues in 95 or 100% ethanol more than a total of 2 hours or the tissues will
harden. Tissues can be stored in 70% ethanol at any time during an interruption in the routine.
II. CLEARING
A. Clearing Agents
There are many clearing agents in use. often the choice depends on the whim of the technician. it
is desirable to use an agent that does not harden the tissue, that clears properly, and that is miscible
with embedding paraffin as well as with ethanol. The clearing agent serves as a transition medium
between two immiscible compounds, ethanol and paraffin.
8. Xylene Commonly used, tends to harden tissue if left in too long, neurotoxic (hangover!)
9. Benzene or toluene. Commonly used; clears overnight.
10. Cedarwood oil. Slightly slower in penetrating than benzene is; does not cause hardening;
does not interfere too seriously with paraffin penetration if it is not completely removed.
1. 100% ethanol: cedarwood oil (1:1), 1 to 2 hours.
2. Fresh cedarwood oil; overnight or longer to clear.
3. Tissues can be left in cedarwood oil indefinitely. It does not harden the tissue.
11. Methyl benzoate.Very good for clearing; does not harden tissues; avoids use of xylene;
benzene is used to rinse out the clearing agent. It penetrates almost as fast as does
cedarwood oil (12 to 24 hours) and is most valuable with the Peterfi celloidin-
impregnation technique.
12. Dioxane.Many directions and techniques make use of dioxane, which is miscible both
with water and paraffin. It is used primarily when time is important because the tissues
may be embedded with paraffin within 4 hours after fixation. The tissues are transferred
to dioxane straight from Bouin's fluid or a formalin fixative. The dioxane is changed 3
times within 4 hours and the tissues are transferred directly to paraffin (3 changes are
made in a total of 90 minutes). Dioxane causes greater shrinkage than xylene does. In
addition, it is dangerous. Fumes of dioxane are toxic to humans; reportedly it is liver
poison. Dioxane must be used in a hood at all times and must be stored in tightly sealed
jars.
III. INFILTRATION
Never leave a tissue in the paraffin oven for more than 4 hours. The shorter the time in the hot
oven with adequate paraffin impregnation and evaporation of clearing agent, the better for the
tissue. Tissues become increasingly harder and more brittle as they are heated. Generally, use
Paraplast with a melting point of 56 to 58 degrees Celsius.
During the winter 54 to 56 degrees Celsius Paraplast may be used if the tissue is cut in a
cool room.
During the summer it may be necessary to use 60 to 63 degrees celsius Paraplast. This is
to be avoided if possible in order to not to "cook" the tissue. "Cooked" tissue does not
section or if it does, it does not stain well and most details are destroyed.
Bioloid paraffin (a mixture of paraffin and other waxes) and Tissuemat are also used
IV. EMBEDDING
The tissue is now going to be placed in a mold and cooled. The choice of mold will depend on the
type of chuck in the microtome you will use to section the tissue. Stainless steel, ceramic, paper,
plastic, and aluminum foil molds can be used. The basic method is the same for each.
13. Spray a stainless steel mold with releasing compound then pour a small amount melted
Paraplast (fresh) into it. This will be demonstrated.
14. Transfer the tissue with hot forceps and orient it properly in the center of the depression.
15. Place the white plastic form on top of the mold and fill with melted paraplast. Be sure
that the form is labeled. If a box or dish method is used insert a paper label to mark the
point of orientation.
16. Remove from heat.
17. Cool the top surface of the Paraplast by blowing gently on it. Tissues at this stage are
very brittle; handle
18. them with care. As soon as a scum of Paraplast has formed on top, sink the cup gently
into a cold water bath or place it on a refrigerated surface.
19. The block will be ruined if it is submerged before its upper surface has formed a
protective scum. Cool thoroughly in cold running
20. tap water. If you use ice water for the final cooling, you may split the block owing to too
rapid shrinkage. Paraplast naturally splits in the line of least resistance-right through the
tissue. If you use plastic cups, the Paraplast block can be removed as soon as it is cooled.
The stainless steel mold should slip off easily when cool and can be used again.
Orientation of tissues in the Paraplast block is important for tissues such as duodenum when
sections in a predetermined plane, such as cross sections, are required. Also, trimming is
excessively difficult in a block embedded with two or more tissues if they are not carefully lined
up before the Paraplast is cooled.
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