Clearing, Infiltration,

and Embedding
Maricelle D. Manlutac, RMT, MPH, MLS(ASCPi)
Faculty, University of Santo Tomas
Examination of Fixed Tissues:
Histopathologic Techniques/Steps

1.Numbering 7. Blocking

2. Fixation 8. Trimming

**Decalcification 9. Sectioning

3. Dehydration 10. Staining

4. Clearing 11. Mounting

5. Impregnation 12. Labeling

6. Embedding
Clearing / Dealcoholization
 Process of removing alcohol from tissue section

 After staining – transparent tissues

 High refractive index
Clearing Agents:
 Miscible with alcohol and paraffin &/or mounting
medium

 Should not produce excessive tissue shrinkage &

hardening

 Should not evaporate quickly in a waterbath

 Should make tissues transparent

Clearing Agents:

 Benzene  Oil of Wintergreen

 Chloroform  Carbon Disulfide
 Xylene  Carbon Tetrachloride
 Toluene  Carbol-xylene
 Cedar Oil  Tetrahydrofuran
 Clove Oil  Terpineol
 Oil of Bergamont  Phenol
 Oil of Origanum  High Test “Aviation Lead Free
Gasoline”
Benzene
 Routine work: Rapid acting

 Volatilizes rapidly in paraffin oven

 Tissue shrinkage if left for a long time

 Carcinogenic aplastic anemia

Chloroform
 For tough tissues – decalcified, nervous, lymph
nodes, embyros – minimum shrinkage and
hardening

 For large specimens

 Prolonged inhalation Toxic to liver

 Does not make tissues transparent

 Tissues tend to float

Xylene (Xylol)
 Colorless: most common

 Most rapid: Clearing time is 15-30 minutes

 Cheap

 Milky if incomplete dehydration

 Hardens/shrinks tissues – not for nervous system & lymph

nodes

 Hard/brittle tissues if > 3 hours

Toluene
 Expensive - Slower than xylene & benzene

 Not carcinogenic; but will emit fumes that are toxic

upon prolonged exposure

 Tissues do not become excessively hard and brittle

even if left here for 24 hours
Cedar Oil
 Very penetrating: “best” clearing agent

 Very expensive: Extremely slow

 For CNS, smooth muscles, skin, and cytological

work

 Milky on prolonged storage (filter)

 May produce crystals – heat to 200 °C

Clove Oil
 For skin and smooth muscle

 Wax impregnation here – slow / difficult

 Tissues become brittle use OIL of BERGAMOT

 expensive
Aniline Oil
 For clearing embryos, insects and delicate
specimens
Oil of Origanum
 Spanish Hop Oil or Thyme Oil

 For Skin
Oil of Wintergreen
 Methyl salicylate

 Artificial Oil

 For Delicate tissues : avoid tissue hardening

Carbon Disulfide
 For Smooth muscles

Disadvantage:
 Foul odor
 Wax impregnation is slow
Carbon Tetrachloride (CCl4)
 Like chloroform

 Hepatotoxic
Carbol-xylene
 Anhydrous crystals of phenol to xylene

 For Friable tissues

 Corrosive
THF
 Colorless liquid

 BP: 65°C ; FP: -108.5°C

 For cellular organs, friable tissues, skin,

athermatous arteries, and hard collagenous
specimen

 Dehydrates and clears

 Non toxic but with offensive odor

Terpineol
 Expensive

 1 part xylene and 4 parts terpineol

 For delicate material: Eyes

Phenol
 Good for paraffin and celloidin work

 Dehydrates and clears very quickly

 For smooth muscles

High Test “Aviation Lead Free Gasoline”

 Cedar oil, gasoline, petroleum ether

 Excellent clearing agent

 Least hardening of tissues

Impregnation / Infiltration
 Process whereby the clearing agent is completely
removed from the tissue
 Clearing agent is replaced by a medium that will fill tissue
cavities
 To give firm consistency to the specimen
 Allow easy handling and cutting thin tissue sections
Types of Tissue
Impregnation/Embedding Media:
 Paraffin Wax
 Manual – 4 changes of wax 15 mins interval
 Automatic – Autotechnicon, Elliott Bench type
 Vacuum – fastest, negative atmospheric pressure

 Celloidin (Collodion)
 Large hollow cavities which tend to collapse
 Large tissue sections: whole embryo
 Hard and dense tissues: bones and teeth

 Gelatin
 Histochemical and enzyme studies
 Does not require dehydration and clearing

 *Plastic
 Epoxy, polyester, or acrylic
Embedding
 Casting / Blocking

 Place tissue in a precisely

arranged position in a mold with
a medium which is then allowed
to solidify

 Orientation
• Precise position on the mold
before embedding
• Microtome before cutting
• Slide before staining
Molds for Embedding
 Leuckhart’s Embedding Mold
 Compound Embedding Unit
 Plastic Embedding Rings & Base Mold
 Tissue Tek – machine w/ warm plate

 Disposable
 Peel Away
 Plastic Ice Trays
 Paper boats
Molds for Embedding

b. Leuckhart’s Embedding
a. Stainless Base Mold
Mold

d. Compound Embedding
c. Plastic Embedding Rings
Unit
Methods of Embedding
 Paraffin Method of Embedding
 Simplest; most common

 Celloidin Method of Embedding

 Hard or friable specimen, neurologic works

 Gelatin Method of Embedding

 Friable, delicate, and tissue fragment section requiring Frozen
section
 Specimen that does not require dehydration and clearing
Methods of Embedding
 Vacuum Embedding
 Dense and difficult to impregnate
 Internal temperature chamber: 2-4 °C above MP of wax

 Double Embedding Method

 Tissues first infiltrated with Celloidin Embedded in Paraffin
 Large dense firm tissues: Brain

 Plastic Embedding
 Ultra-thin sections for EM