HISTOLOGY LABORATORY HANDOUTS

Histology & General Histological Techniques II

Dehydration, Clearing, Infiltration and Embedding
See also other Histological Techniques
Animal Dissection and Fixation of Tissues
Tissue Processing: Sectioning And Slide Mounting
Stains and Staining
A. GENERAL PARAFFIN PROCESSING ROUTINE

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.

Alcohol Protocol Dioxane Protocol

Alcohol solution Time -- In tissue rotator underhood
95% Alcohol .5 to 1 hours Dioxane solution Time
95% Alcohol .5 to 1 hours Dioxane I 15 minutes
100% Alcohol .5 to 1 hours Dioxane II 15 minutes
100% Alcohol .5 to 1 hours Dioxane III 15 minutes

Clearing Method: Infiltration:No Longer Than 4 Hours

Clearing solution Time Paraplast changes Time
Alcohol /Xylol 1:1 15 minutes 35o Celsius 30 minutes
Xylol I 15 minutes Paraplast I 30 minutes
Xylol II 15 minutes Paraplast II 60 minutes
Xylol /Paraplast 1:1 15 minutes Paraplast III

II. Protocol
We will use the following schedule to process our tissue. No Times are listed. Please fill in

Start __________________________ and finish during Laboratory in the afternoon

Dehydration Clearing Infiltration:

Change to Time Change to Time Change to Time
95% Alcohol _________ Alcohol /Xylol 1:1 _________ Paraplast I* _________
95% Alcohol _________ Xylol I _________ Paraplast II* _________
100% Alcohol _________ Xylol II _________ Paraplast III* _________
100% Alcohol _________ Xylol /Paraplast 1:1* _________
 Oven temperature at * 35o or the lowest remperature for the melting point of the paraplast being
used. Note: the higher the melting point the harder the paraplast. The higher the melting point the
greater possibility you may "cook" your tissue!!

Embedding at _____________________ PM

Embedding: Will be Demonstrated

1. Spray a stainless steel mold with releasing compound

2. Pour a small amount melted Paraplast (fresh) into it.
3. Transfer the tissue with hot forceps and orient it properly in the center of the depression.
4. Place the white plastic form on top of the mold and fill with melted paraplast.
5. Remove from heat. Cool the top surface of the Paraplast by blowing gently on it.
6. As soon as a scum of Paraplast has formed on top, sink the cup gently into a cold water
bath.
7. Cool thoroughly in cold running tap water.

B. GENERAL PARAFFIN PROCESSING INFORMATION

A. General Dehydrating Protocol

A standard graded series of alcohols are used most frequently to dehydrate or hydrate tissues to
prevent shrinkage or distortion of the cells. This is used when one immiscible solvent must be
replaced by another. The following represents standard dehydration procedures.

Dehydration Dehydration After Staining Slides

in Preparation for Embedding in Preparation for Coversliping
Aqueous fixative Aqueous stain
Water Water (rinse)
35% Ethanol 35% Ethanol
50% Ethanol 50% Ethanol
70% Ethanol Storage 70% Ethanol Storage
70% Ethanol + Lithium carbonate 95% Ethanol
treatment for picric acid 95% Ethanol
95% Ethanol from Alcoholic fixative (+ eosin for counter stain if used.)
95% Ethanol (+0.5% eosin for tiny, 100% Ethanol
transparent objects.) 100% Ethanol
100% Ethanol CLEARING resin medium
100% Ethanol
CLEARING Paraffin or other
Embedding medium

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

1. After Clearing Transfer to a mixture of xylene (benzene) saturated with

Paraplast (paraffin and plastic polymers) chips. On top of
2. the paraffin oven or in some warm spot at about 35 degrees Celsius, for 15
minutes.
3. Infiltrate with hot paraplast in a 58 degree celsius paraffin oven in an open
container.
4. For 3 changes with the following time sequence: 30 minutes, 30 minutes, 1 hour.

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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