Infiltration & Embedding

Histology

What are the advantages of paraffin?

    Large number of blocks may be processed in a short period of time.

    Serial sections can easily be obtained.

    Routine and special stains can be done easily.

    It is more cost effective than other embedding media.

What does the basic properties of paraffin vary with?

The melting points

What are the advantages of paraffin?

    Large number of blocks may be processed in a short period of time.

    Serial sections can easily be obtained.

    Routine and special stains can be done easily.

    It is more cost effective than other embedding media.

What does the basic properties of paraffin vary with?

The melting points

What happens with higher melting point paraffin?

The paraffin is harder, provides better tissue support, and thin sections can be obtained, however, monitoring may be different

What happen with lower melting point paraffin?

The wax is softer, provides less support for harder tissues, and thin sections are more difficult to get but ribboning is easier

What criteria should the type of paraffin wax match?

The paraffin type should match the tissue to be sectioned, temperature of sectioning, and the staining technique involved

How much over the melting point should the paraffin be kept at

2-4 degrees celsius

What is the most common paraffin melting point range

55-58

What temperature is paraffin kept at

60 degrees C

What type of stains are affected by increased melting points

Immunohistochemical stains

What is sectioning quality affected by?

Overheating for prolonged periods can lead to poor sectioning quality, affecting the integrity of the tissue samples. 10-20 degrees over melting point

Why are paraffin wax additives used?

To modify the consistency and change its melting point, increase hardness for cutting thinner sections or sections at high ambient temperature, increase hardness for support, increase stickiness for ribbons, alter crystalline structure of paraffin, reduction in crystal size improve sectioning properties, greater support to the tissue owing to better fit of crystals within the tissue matrix, harder tissue may be sectioned and thinner sections produce

What does beeswax do?

Reduce crystal size, increase stickiness and adhesion

What do other waxes do?

Produce smoother textures and reduces crystal size

What does rubber additive do?

Facilitate easier ribboning, reduce brittleness, increase stickiness

What does plastic addtives do?

Increase hardness and support

What are the considerations of paraffin processing?

-When the paraffin becomes yellow and soap-like it means that the crystalline structure has changed due to oxidation

-The tissue should remain in paraffin at the shortest time necessary for good infiltration

-Prolonged heat will cause shrinkage and hardening, as liquid paraffin congeals quickly after heat is removed and as paraffin solidifies it becomes more difficult to orient the tissues and to ensure that all pieces are at the same level

-The infiltration media needs to be free of clearing agents and therefore 3 changes of paraffin are suggested

-Paraffin is greatly aided by a vacuum

-Duration of paraffin depends on thickness and texture of tissue and the usual infiltration time is 2-3 hours of a processing cycle

-Proper quality control must be ensured by recording paraffin temperature on a daily basis and rotating and changes of paraffin should be done frequently

-Paraffin blocks with exposes tissue must be dipped lightly in molten paraffin to seal exposed tissue

What are the different mold shapes?

Large pangs, paper boats, and L-shaped

Are they many different sizes of molds?

yes

What is the purpose of the mold?

To give support and shape the molten paraffin while it solidifies and the solid must be easy to remove from the mold after use

What are molds made from?

Aluminum allow, stainless steel, plastic, copper plates

Why can substitution infiltration media used?

-Impregnation not hard and fails to provide enough support

-Tissue adversely affected by heat

-Dehydrants and clearing agents may destroy or distort tissues or components

-Adhesion between paraffin and tissue is inadequate resulting in break away of tissue during sectioning

-Crystalline structure of paraffin not suitable requiring an amorphous medium

-Sectioning cannot be cut thin enough

What is an example of a water soluble wax (carbowax)?

Solid water soluble polyethylene glycols

Describe the characteristics of a carbowax and how they act.

They do not need to undergo dehydration and clearing as they can infiltrate from aqueous fixative

fat will not dissolve but carbowax will not infiltrate extremely fatty tissues

Takes place in a period of 3 hours with 3 changes

Some enzymes remain active and can be demonstrated

Softer than paraffin

Sections will dissolve in a water bath therefore a substitute should be used

Should be cut and chilled from refrigerator

Impregnation of brain and spinal chord is very slow

Describe celloidin-nitrocellulose

Amphorous, slightly yellowish substance, completely insoluble in water and is dissolved in one of two 50:50 mixtures of either diethyl ether: ethanol or diethyl ether: methanol. Gives superb distortion free sections with normal shrinkage for nervous system specimen, chemicals are hazardous and explosive and very time consuming procedure weeks to months

Explain the processing cycle of celloidin-nitrocellulose

It requires no heat, begins with dehydration with 95% and absolute alcohol, treatment of section with equal parts of absolute alcohol and ether, infiltration is done by graded solutions and the time depends on tissue size, usually ending up in a solution which contains 12% cellulose

Explain embedding with celloidin-nitrocellulose

Embedding will take place in solutions containing up to 14% celloidin.

Embedding dish must be air tight.

Tissue must be left undesirable for several days in order to let trapped air escape.

This must be done slowly so that no air bubbles form.

Air bubble could lead to problems during sectioning.

Chloroform can be used to harden the embedding media.

Explain sectioning with celloidin-nitrocellulose

Sections embedded with celloidin-nitrocellulose can be cut wet or dry.

Wet : 80% alcohol

Dry: cedar wood oil

Cut sections must be kept in 80% alcohol prior to staining.

Staining:

Done by hand, by transferring the section from solution to solution with a glass rod.

Mounting on the slide is the last step.

Explain how glycol methacrylate is used

An acrylic resin that is miscible with water.

Infiltration takes place after dehydration of tissue with 95% alcohol.

Provides good support for very hard tissue, such as undecalcified bone.

Allows 1-2µ sections to be cut.

Glass knives must be used.

Used for kidney, liver, lymph node and bone marrow biopsies.

Sectioning is more difficult and usually requires a special microtome.

Cellular detail is well preserved and distortion is minimal

Staining can prove to be more difficult depending upon the stain.

Chemicals are hazardous, sensitizers and irritants must be used under a hood.

What are the common epoxy resins?

Araldite, epon, spur

What does the final embedding mixture of epoxy resins contain?

epoxy resins, hardeners, and catalysts with polymerization done at 60º.

Needs a transition fluid, usually propylene oxide since the epoxy resin is not miscible with alcohol.

What is epoxy resins used for

Used primarily for electron microscopy, or ultrastructural examination of tissue.

What size should epoxy resins sections be?

80-90nm

What are epoxy resins sectioned with?

a diamond knife

what is dangerous about epoxy resins?

Resins are sensitizers and irritants and can cause dermatitis, therefore skin contact should be avoided.

Describe agar as a substitution infiltration medium

Does not provide enough support for sectioning, but can be used to help produce a single block of friable tissue.

Can assure proper orientation during embedding.

Describe gelatin as a substitution infiltration medium.

Has a lower melting point than agar.

Less satisfactory for double embedding.

Describe OCT embedding

This is a water soluble embedding media, that leaves no residue on the slide.

Primarily used for frozen section specimens, to give the tissue support.

Used when there is the need to:

Freeze fast

Cut thin sections

Support the tissue