Laboratory #6 Infiltration and Embedding

Impregantion

Process whereby the clearing agent is completely removed from the tissue and replaced by a medium that will fi ll the tissue cavities and give it a fi rm consistency.

easier handling and cutting

Impregnation allows for ? into thin sections.

Impregantion

Process whereby the clearing agent is completely removed from the tissue and replaced by a medium that will fi ll the tissue cavities and give it a fi rm consistency.

easier handling and cutting

Impregnation allows for ? into thin sections.

Embedding

This is also known as "casting" or "blocking" the process by which impregnated tissue is placed into a precisely arranged position in a mold containing a medium which is then allowed to solidify.

well-infiltrated tissue

The specimen used in embedding is?

Parrafin Wax

Simplest, most common, best embedding medium for routine use.

56-60C

Paraffin wax - Melting:

-5º – 10ºC / cold water

Paraffin wax - Cool rapidly (refrigerator):

indefinitely

Paraffin wax: Advantages

• tissue blocks and unstained mounted sections can be stored ?

RNA and DNA

Paraffin wax: Advantages

• ? may be recovered decades after

shrinkage and hardening

Paraffin wax: Disadvantages

• prolonged impregnation will cause excessive tissue ?, making cutting difficult

fatty tissues

Paraffin wax: Disadvantages

• not recommended for ?

Celloidin

Suitable for tissues with large, hollow cavities which tend to collapse.

thin (2%), medium (4%) or thick (8%)

Celloidin is supplied in ? solutions of cellulose dissolved in equal parts of ether and alcohol.

Neurological tissues

Celloidin is recommended for:

Gelatin

Rarely used except for when dehydration is to be avoided; does not need dehydration.

delicate specimens and frozen tissue sections

Gelatin is used as an embedding medium for:

water-soluble; dehydration and clearing

Gelatin is ? and does not require ?

1% Phenol

In gelatin ? is added to prevent molds.

at least 25 times

The volume of the impregnating volume should be ? the volume of the tissue.

Plastic (resin)

Provides superior results for light microscopy, especially for hard tissues like bone and thin sections.

• Epoxy

• Polyester

• Acrylic

Categories for Plastic (Resin):

Epoxy

Are hydrophobic and subsequent oxidation by peroxide to correct this may produce tissue damage. Epoxy plastics vary in infiltration speed.

• Bisphenol A

• Glycerol

• Cyclohexene Dioxide

Types of Epoxy Plastics:

Polyester plastics

Were introduced for electron microscopy in the mid 1950s, but have been superseded by superior epoxides.

Acrylic plastics

Are made up of esters of acrylics or methacrylic acid and are used extensively for lightmicroscoру. Are extremely hydrophilic, allowing for many staining methods, yet are tough enough when dehydrated to section well.

Paraffin Wax Embedding

Polycrystalline mixture of solid hydrocarbons produced during the refi ning of coal and mineral oils.

Paraplast

A paraffin and synthetic polymer mix melting at 56-57ºC, offers superior elasticity and resilience to paraffin wax.

Ester Wax

Harder than paraffin, melts at 46-48ºС, and is insoluble in water but soluble in 95% ethyl alcohol and other clearing agents like Cellosolve or xylene.

Water soluble wax (carbowax)

Improves paraffin wax blends, increasing adhesion, hardness, and plasticity

Leuckhart’s Embedding Mold

With two adjustable L-shaped metal strips on a plate, creates even blocks with parallel sides and a well shaped wax setting.

• Leuckharts Embedding Mold

• Compound Embedding Unit

• Plastic Embedding Rings and base Molds

• Disposable Embedding Molds

Blocking Out Molds:

• Peel Away

• Plastic Ice Trays

• Paper Boat

Disposable Embedding Molds

Peel Away

Disposable plastic embedding molds, in three sizes. Easily removed after wax solidification, creating perfectly even blocks needing no coarse trimming.

Plastic Ice Trays

Busy laboratories may benefit from using ordinary refrigerator-type embedding molds Each compartment holds one tissue block, removable after wax solidification by bending or pretreating the mold with glycerin or liquid paraffin.

Paper Boats

Inexpensive and simple to create, serving as effective embedding tools for both celloidin and paraffin wax blocks

across the block

Elongated tissues are placed:

Tissues with lumen are embedded on end so that the lumen is visible.

on edge

Tissues from "hollow organs" should be embedded ? to show the various layers.

right angle to the surface

Tissues with an epithelial surface are embedded to provide sections in a plane at a ?.

diagonally

Hard tissues will section more easily if it is embedded ?

aligned across the long axis of the mold

Multiple tissues are ?, and not placed at random.

toward the center

Fragments are clustered ? of the block.

Orientation

The process by which the tissue is arranged in a precise position in the mold during embedding, on the microtome before cutting, and on the slide before staining.

All layers in transverse section

Orientation for Tubular tissue:

All layers should come

Orientation for Skin:

Keep in Center

Orientation for Endometrial curetting:

Keep diagonally

Orientation for Long tissue:

All layers should come

Orientation for Intestine:

Swiss role

Orientation for Membrane