infiltration/impregnation & embedding

Infiltration/Impregnation

Process whereby clearing agent is completely removed from the tissue and replaced by a medium that will fill tissue cavities yielding a firm consistency

1:>25

Tissue: Impregnating Agent

Embedding/Casting/Blocking

Process by which impregnated tissue is placed precisely arranged position in a mold containing the medium allowing it to solidify

Orientation

process in which tissue placement is arranged in the mold, on microtome prior to cutting, and on the slide prior to staining

Paraffin Wax

Simplest and most common medium

56ºC

PARAFFIN WAX

melting point

Rotary Microtome

PARAFFIN WAX

microtome

Celloidin

• Purified form of collodion/nitrocellulose

• Yellowish with rubbery consistency

• For hard and dense tissue (bones and teeth) large sections of whole organs (eyes)

Gelatin

• Used when dehydration is avoided

• Histochemical and Enzyme studies

• Delicate and friable specimen

Epoxy

PLASTIC MEDIUMS

Light Microscopy (LM) and Electron Microscopy (EM)

Acrylic

PLASTIC MEDIUMS

High Resolution Light Microscopy (LM))

Methacrylate

PLASTIC MEDIUMS

Electron Microscopy (EM)

Double Embedding Method

Infiltrated with celloidin and embedded with paraffin to facilitate cutting of large blocks

2-5ºC above M.P (55-60C)

Paraffin Oven maintained at least __

Overheated Paraffin (>60ºC)

Brittleness, Shrinkage, and Hardening of tissue

100-105C

Water interferes with impregnation - water is removed by heating wax to __

Paraplast

SUBSTITUTES OF PARAFFIN WAX

• Mixture of highly purified paraffin and synthetic plastic polymers

• More elastic and resilient

Embeddol

SUBSTITUTES OF PARAFFIN WAX

• Synthetic wax substitute similar to Paraplast

• Ideal for whole eyes

Bioloid

SUBSTITUTES OF PARAFFIN WAX

• Synthetic wax recommended for eyes

Ester Wax

SUBSTITUTES OF PARAFFIN WAX

• Lower M.P. 46-48ºC

• Harder than Paraffin

• section with the use of a Heavy-duty microtome (Sliding/Sledge)

Carbowax

SUBSTITUTES OF PARAFFIN WAX

• Polyethylene glycol

• Miscible and soluble to water

• Do not require dehydration and clearing

• Sections are difficult to to float out and mount

Wet Celloidin

CELLOIDIN METHOD

Recommended for bones, teeth, large brain sections, and whole organs

70-80% Alcohol

CELLOIDIN METHOD - Wet

Tissue blocks stored in __ or else tissue blocks becomes discolored, dry, and shrunken

Dry Celloidin

CELLOIDIN METHOD

Preferred for whole eye section

Gilson’s Mixture

CELLOIDIN METHOD - Dry

• Chloroform and Cedarwood Oil

• added to celloidin blocks prior to hardening to make tissue transparent

TWICE the thickness of the tissue

TECHNIQUES FOR EMBEDDING TISSUES

Depth of the mold should at least be __

10ºC

TECHNIQUES FOR EMBEDDING TISSUES

prevent cracking of the tissue block during solidification of paraffin

Leuckhart’s Embedding Mold

MOLD FOR EMBEDDING

2 L-shaped adjustable to fit the size of block

Compound Embedding Unit

MOLD FOR EMBEDDING

Several compartments embedding more specimen at a time

Plastic Embedding Ring & Base

MOLD FOR EMBEDDING

block is ready; less paraffin

• Peel-A-Away

• Paper Boat

• Plastic Tray

MOLD FOR EMBEDDING

disposable

Watch Glass

MOLD FOR EMBEDDING

Ideal for embedding fragmentary biopsies

Test Tubees

MOLD FOR EMBEDDING

• Small fragment (Bone marrow) which concentrates them without use of forceps

• need to break it to obtain it

Trimming

Removing of excess wax after embedding using a knife or blade

truncated pyramid/4-sided prism

TRIMMING

shape

2 mm

TRIMMING

surrounding the block

Incomplete fixation

Tissue is soft when tissue block is trimmed

Incomplete Dehydration

Clearing agent turns milky

Incomplete Impregnation

Air holes found on tissue during trimming