CLEARING Flashcards
• serves as a bridge between dehydration and infiltration
Clearing
Clearing
• serves as a bridge between _____ and _____
dehydration and infiltration
______ and ______ are immiscible
paraffin wax and ethanol
______ are miscible with both ethanol and paraffin wax
clearing agents
Clearing agent
Xylene
Clearing
many (but not all) clearing agents impart an _____ or _____to the tissue
- high refractive index
optical clarity or transparency
Clearing
• remove substantial amount of____ from tissue
fat
_____presents a barrier to wax infiltration
fat
Clearing
• performed in both:
- embedding process
- mounting tissues
Clearing in
- embedding process
Bridge between dehydration and infiltration
Clearing in - mounting tissues
After staining
To make slide clearer
Choice of
Clearing Agent
• depends on:
- the type of tissue
- type of processing and processor system
- intended processing conditions
• e.g. temperature, vacuum and pressure - safety factors, cost and convenience
Examples of Clearing Agents
Organic Solvents
Oils
Clearing agents
- Xylene
- Benzene
- Toluene
- Terpineol
- Chloroform
- others
Oils
- Cedarwood oil
- Aniline oil
- Clove oil
- Oil of Bergamot
- Oil of oreganum
- lead-free gasolie
Xylene (3) characteristics
• most common
• rapid
• readily miscible with paraffin wax
tissues best cleared from absolute alcohol
Why?
- incomplete dehydration will cause xylene to turn milky
• used for both embedding and mounting procedures
Xylene
Xylene
prolonged treatment is avoided
- can cause…
hardening of tissues
Xylene
- not preferred for ___ or ____
brain or CNS tissues
Procedure
• Xylene 1
• Xylene 2
• Xylene 3
- Place on paper towels to dry.
- Wrap in foil.
20 min
20 min
45 min
Actual lab procedure
X1
X2
30mins
45mins
Initial deparaffinization
Microwave oven
Xylene
Sa H&E staining, ang first 3 stages didto kay…for
Final paraffinization
After staining
Last 3 xylene station is for…
Clearing before mag mount
Aside from clearing agent, cylene can be used as an…
Indicator if dehydration is not done properly
Milky xylene
What to use for brain or CNS tissues
Toluene
Why is toluene not routinely used
Expensive
Reagent which clears tissue slowly
Toluene
In the______ we clear tissues after dehydration to prepare the tissues for its exposure to wax.
embedding process
In______, we clear tissue sections after they have been stained to prepare it for microscopic evaluation.
mounting
: clears less rapidly than xylene or chloroform, but does not harden tissue – hence, it is preferred for processing brain sections
Toluene
Its properties are relatively similar to xylene.
It is non-carcinogenic although high concentrations may emit fumes that are toxic upon prolonged exposure.
It is also more expensive.
Toluene
: more gentle and rapid than xylene and toluene and is probably the best transition solvent, though toxicity and possible carcinogenicity preclude its use in histology.
Benzene
: expensive, heavy, highly volatile, slowly penetrating transition solvent. It causes less brittleness than xylene and is often used on dense tissues such as uterus and muscle which can be cleared overnight without undue hardening.
Chloroform
Since_____ attacks some plastics and sealants its use may be restricted in certain closed system processors.
chloroform
: clear almost colorless mixture of isomers with a faint pleasant odor and very low evaporation rate
Terpineol
It clears tissues from 80%-90% alcohol with minimal hardening. It is a particularly useful substitute for cedarwood oil in manual processing and is also used in open-dish microscopic examination of cleared parasitic helminths.
Terpineol
Tissues may remain in it indefinitely without harm.
However, it is difficult to eliminate from paraffin wax.
Terpineol
Other examples for organic solvents are:
dioxane, esters like amyl acetate, methyl benzoate and methyl salicylate; carbol-xylene and carbon tetrachloride.
: largely composed of cedrene, rapidly clears tissues from 95% alcohol, hardens tissues the least of all the transition solvents, but is difficult to eliminate from tissues during wax infiltration
Cedarwood oil
It is particularly useful for processing dense tissues such as uterus or scirrhous carcinomas, and has a role in forensic histopathology in processing the hardened skin margins of electrical burns and bullet wounds.
Cedarwood oil
Purpose of clearing
Bridge between dehydration and infiltration
Imparts an optical clarity to tissue
Remove substantial amount of fat
Final deparaffinization
