CHAPTER 2 CHAPTER 3

FRESH TISSUES EXAMINATION FIXATION AND FIXATIVES

-vary according to structural and chemical components of Fixation – fixing or preserving fresh tissue for examination
the cells to be studied
-may be done on fresh/preserved tissue - Quality of section on the slide is only as good as
the quality of the fixed specimen
METHODS
1. Teasing/Dissociation Primary aim: preserve the morphological and chemical
- selected tissue is immersed in watch glass integrity of the cell in as life-like manner.
containing NSS then carefully dissected/separated and - Shape, structure, intercellular relationship and
examined under the microscope. chemical constituents of tissues are
preserved.
2. Squash Preparation/Crushing - Prevents degeneration, decomposition,
- small pieces not more than 1 mm are placed in a slide putrefaction, and distortion of tissues
and forcibly compressed with another slide or with a after removal from the body.
cover glass.
Secondary goal: harden and protect the tissue from the
3. Smear Preparation trauma of further handling
- examining sections of sediments, whereby cellular
materials are spread lightly over a slide by means of wire Stabilizing proteins: most important reactions for
loop or applicator. maintaining morphology in the fixation of tissues for
routine histopathology
Streaking – zigzag line - They are fixed to structural proteins and
Spreading – teasing mucous strands thus rendered insoluble
Pull-Apart – two slides are used
Touch Preparation – cells are transferred to the slide 1. To preserve the tissue
2. To prevent breakdown of cellular elements
4. Frozen Section – Tissue is frozen with liquid nitrogen 3. To coagulate or precipitate
and a section is examined under the microscope. protoplasmic substances

PROCESSING OF TISSUES Additive fixation – fixative becomes part of the tissue

Fixation Negative fixation – fixative does not incorporate into the
Dehydration tissue but alters the tissue composition and stabilizes the
Clearing tissue by removing the bound water attached.
Infiltration (Impregnation)
Embedding
Trimming MAIN FACTORS INVOLVED IN FIXATION:
Section-Cutting 1. Hydrogen Ion Concentration – pH 6 and 8
Staining 2. Temperature – Formalin heated at 60C
Mounting 3. Thickness of section – 2cm2 for light microscopy
Labelling 4. Osmolality – slightly hypertonic
5. Concentration – low conc. of glutaraldehyde
6. Duration of fixation – 2-6 h in buffered formalin

PRACTICAL CONSIDERATIONS OF FIXATION:

1. Speed
2. Penetration
3. Volume
4. Duration of Fixation
EFFECT OF FIXATIVES - increase optical differentiation of cells and tissues
- harden soft and friable tissues - act as mordants or accentuators
- make the cells resistant to damage and distortion - reduce the risk of infection
- inhibit bacterial decomposition
CHARACTERISTICS OF A GOOD FIXATIVE 2. According to Action
1. Cheap a. Microanatomical Fixatives – permits the
2. Stable general microscopic study of tissue structures
3. Safe to handle 1. 10% Formol Saline
4. Kills the cell quickly producing minimum 2. 10% Neutral Bufered Formalin
distortion of cell constituents. 3. Heidenhain’s Susa
5. Inhibit bacterial decomposition 4. Formol sublimate
6. Produce minimum shrinkage of tissues 5. Zenker’s solution
7. Harden tissues making cutting sections easier 6. Zenker formol
8. Isotonic, causing minimal physical and 7. Ouin’s solution
chemical alteration of the cells and their 8. Brasil’s solution
constituents.
9. Make cellular components insoluble to b. Cytological – preserve the specific parts and
hypotonic solutions particular microscopic elements of the cell
itself

TYPES OF FIXATIVES 1. Nuclear Fixative – preserve nuclear

1. According to composition structures Flemming’s fluid
a. Simple Fixative – made up of only Carnoy’s fluid
one component substance. Bouin’s fluid
i. Aldehydes Newcomer’s fluid
1. Formaldehyde Heidenhain’s Susa
2. Glutaraldehyde
ii. Metallic Fixatives 2. Cytological Fixatives – preserves
1. Mercuric Chloride cytoplasmic structures
2. Chromate Fixatives Flemming’s fluid without acetic acid
a. Potassium Kelly’s fluid
dichromate Formalin with “post-
b. Chromic acid chroming” Regaud’s fluid
3. Lead Fixatives (Muller’s fluid) Orth’s fluid
a. Picric Acid
b. Acetic Acid 3. Histochemical Fixatives – preserve
c. Acetone chemical contents of cells and tissues
d. Alcohol Formol Saline 10%
e. Osmium Absolute Ethyl Alcohol
tetraoxide Acetone
4. Heat Newcomer’s Fluid

b. Compound Fixative – made up of two LIPID FIXATION

or more fixatives – Use mercuric chloride and potassium dichromate
- Baker’s formol calcium for phospholipids

CARBOHYDRATE FIXATION – Alcoholic formaldehyde

PROTEIN FIXATION – Neutral buffered formol saline or
formaldehyde
GLYCOGEN FIXATION – Rossman’s fluid or absolute alcohol
ALDEHYDE FIXATIVES – fixation of CNS Tissues and General post-mortem tissues
Formaldehyde – widely used (10% formalin) - preserves enzymes and proteins
- DA: fumes are irritating to the nose and eyes
- prolonged storage may induce precipitation of 10% Neutral Buffered Formalin/Phosphate-Buffered
white paraformaldehyde Formalin
- Removal of precipitate is addition of 10% methanol - Sodium dihydrogen phosphate + Disodium hydrogen
phosphate + 40%Formaldehyde + Distilled water
10% Formol-Saline - preservation of surgical, post-mortem and research
- 40% Formaldehyde + NaCl + Distilled water specimens
- best fixative for iron-containing tissues METALLIC FIXATIVES
1. MERCURIC CHLORIDE
Formol-Corrosive (Formol Sublimate) - Mercuric Chloride + Potassium Dichromate +
- Sat. Aq. Mercuric Chloride + 40% Formaldehyde Sodium Sulfate + Distilled Water
- routine post-mortem tissues - most common metallic fixative
- excellent in silver reticulum methods - Tissues fixed with mixtures containing mercuric
- fixes lipids, especially neutral fats and phospholipids chloride (except Susa) contain black precipitates of
mercury.
Alcoholic Formalin (Gendre’s Fixative) -Routine fixative of choice for preservation of cell detail in
- 95% Ethyl Alcohol saturated with picric acid + Strong tissue photography.
formaldehyde solution + glacial acetic acid - Renal tissues, Fibrin, Connective tissues and muscles
- immunoperoxidase studies on tissues - Black deposits may be removed by adding
- used for rapid diagnosis saturated iodine solution in 96% alcohol, the iodine
- good for preservation of glycogen and for being decolorized with absolute alcohol in the
micro- incineration subsequent stages of dehydration.
-used to fix sputum, since it coagulate mucus
Zenker’s Fluid
Glutaraldehyde - Mercuric Chloride + Glacial Acetic Acid
-two formaldehyde residues linked by 3C chains - fixing small pieces of liver, spleen, connective tissue and
-used for enzyme histochemistry and electron microscopy nuclei
-preserves plasma proteins - may act as mordant
- Mercuric deposits may removed by immersing tissues
in alcoholic iodine solution. “de-zenkerization”

Zenker-formol (Helly’s solution)

- Mercuric chloride + Potassium dichromate + Sodium
sulphate + Distilled water + Strong formaldehyde
(40%)
- fixative for pituitary gland, bone marrow and
blood containing organs such as spleen and liver.
- preserves cytoplasmic granules
- Brown pigments are produced if tissues are allowed
to stay for more than 24 hours.
- Pigments can be removed by immersing the tissue
in saturated alcoholic pricric acid or sodium hydroxide

Heidenhain’s Susa Solution

- Mercuric chloride + Sodium chloride + Trichloroacetic
acid + Glacial Acetic Acid + Formaldehyde (40%) +
Distilled water
- tumor biopsies especially of the skin
- excellent cytologic fixative
-Mercuric chloride deposits may be removed by
immersion on alcoholic iodine solution
- the tissue should be transferred directly to a high-
grade alcohol, to avoid undue swelling of tissues caused
by treatment with low-grade alcohol or water.

B-5 Fixative
- Distilled water + Mercuric Chloride + Sodium acetate
- commonly used for bone marrow biopsies
CHROMATE FIXATIVES preserves carbohydrates.
Chromic Acid - Strong oxidizing agent, strong reducing agent must be
- used in 1-2% aqueous solution added.
- precipitates all proteins and adequately
 Potassium Dichromate GLACIAL ACETIC ACID
- used in 3% aqueous solution - Precipitates chromosomes and chromatin materials
- preserves lipids - Essential constituent of most common
- preserves mitochondria compound nuclear fixatives

Regard’s (Muller’s) Fluid ALCOHOLIC FIXATIVES

- Potassium dichromate + Strong formaldehyde 40% - Ideal for small tissue fragments
- Demonstration of chromatin, mitochondria, mitotic - Used as a fixative and dehydrating agent
figures, Golgi bodies, RBC and colloid-containing
tissues Methyl Alcohol - fixing dry and wet smears, blood smears
- Prolonged fixation gives out black deposits and can and bone marrow tissues
be removed by running tap water.
Isopropyl Alcohol 95% - fixing touch preparations
Orth’s Fluid Ethyl Alcohol – blood, tissue films and smears
- study of early degenerative proceses and tissue necrosis Carnoy’s Fluid
- demonstrates rickettsiae and other bacteria - Absolute alcohol + Chloroform + Glacial acetic acid
- preserves myelin - fixing chromosomes, lymph node glands and
urgent biopsies
LEAD FIXATIVES -fix brain tissue for diagnosis of rabies
- used in 4% aqueous solution
- recommended for acid mucopolysaccharides Newcomer’s Fluid
- fixes connective tissue mucin - Isopropyl alcohol + Propionic acid + Petroleum ether
- forms insoluble lead carbonate due to prolonged + Acetone + Dioxane
standing which can be removed by filtration or - fixing mucopolysaccharides and other proteins
adding acetic acid drop by drop
OSMIUM TETRAOXIDE (OSMIC ACID)
PICRIC ACID FIXATIVES - Pale yellow powder which dissolves in water to
- Yellow color may be removed by treatment with form strong oxidizing solution.
another acid dye or lithium carbonate -fixes conjugated-fats and lipids permanently by making
- excellent fixative for glycogen demonstration them insoluble during subsequent treatment with alcohol
- suitable for Aniline stains and xylene
-helps preserve cytoplasmic structure
Bouin’s Solution -fixes myelin and peripheral nerves well
- Sat. Solution of picric acid + Strong formaldehyde 40% -fixes materials for ultrathin sectioning in electron
+ Glacial acetic acid microscopy, since it rapidly fixes small pieces of tissues
- Fixation of embryos and pituitary biopsies and aids in their staining
- Preserving soft and delicate structures -black osmic oxide crystals may be dissolved in cold water
(endometrial curettings) -Osmic acid-fixed tissues must be washed in running water
-yellow stain is useful for handling fragmentary biopsies. for at least 24 hours to prevent formation of artefacts
- collagen, elastic connective tissues
Flemming’s Solution
Brasil’s Alcoholic Picroformal Fixative - common chrome-osmium acetic acid fixative used
- Formaldehyde + Picric Acid + Ethanol/Isopropyl Alcohol - Recommended for nuclear preparation of such sections
+ Tricbloroacetic acid - Aqueous chromic acid 1% + Aqueous osmium
- Fixative for glycogen tetraoxide 2% + Glacial acetic acid
- Less messy than Bouin’s solution -an excellent fixative for nuclear structures
-permanently fixes fat

Flemming’s solution without acetic acid

- Made up only of chromic and osmic acid
- Removal of acetic acid from the formula serves
to improve the cytoplasmic detail of the cell
TRICHLOROACETIC ACID -marked swelling effect on tissues serves to counteract
- incorporated into compound fixatives shrinkage produced by other fixatives
-precipitates proteins -may be used as a weak decalcifying agent
 Factors that affect Fixation of the Tissues
ACETONE A.RETARDED BY:
- Used at a cold temperature ranging from 5*C to 4*C 1. Size and thickness of the tissue specimen-largest
- Recommended for the study of water diffusible tissues require more fixatives and longer fixation time
enzymes especially phosphatases and lipases 2. Presence of Mucus-prevents complete penetration of
- Used in fixing brain tissues for diagnosis of rabies fixative; hence, tissues that contain mucus are fixed
slowly and poorly.Excess mucus may be washed away
HEAT FIXATION with normal saline solution.
- Involves thermal coagulation of tissue protein for 3. Presence of fat- fatty tissues should be cut in
rapid diagnosis thin sections and fixed longer.
- Employed for frozen tissue sections and 4. Presence of blood- tissues containing large amount
bacteriologic smears of blood should be flushed out with saline before fixing
- Destroys RBC 5. Cold temperature- inactivates enzymes
- Dissolves starch and glycogen
B.ENHANCED BY:
SECONDARY FIXATION 1. Size and thickness of tissues- smaller and thinner
- Process of replacing an already fixed tissue in a tissues requires less fixative and shorter fixation
second fixative order time
- Usually with 10% formalin or 10% formol saline 2. Agitation- fixation is accelerated when automatic
as primary fixative or mechanical tissue processing is used.

POST CHROMATIZATION CHAPTER 5

-form of secondary fixation whereby a primarily fixed DEHYDRATION
tissue is placed in aqueous solution of 2.5- 3 % potassium - Process of removing intercellular and extracellular
dichromate for 24 hours to act as mordant for better water from the tissue following fixation and prior to wax
staining effects impregnation

WASHING-OUT Dehydration Agents - Solutions utilized

-process of removing excess fixative from the tissue after General Rule: Whatever the dehydrating agent is used, the
fixation in order to improve staining and remove artefacts amount in each stage should not be less than 10 times the
from the tissues volume of the tissue in order to ensure complete
penetration of the tissue by the dehydrating solution.
1. Tap water- removes:-excess chromates from tissues
fixed in Kelly’s, Zenker’s, and Flemmings solutions Characteristics of Ideal Dehydrating Agent
-excess formalin -excess osmic acid 1. Should dehydrate rapidly
2. 50-70% alcohol – used to wash out excess amount 2. Should not evaporate very fast
of picric acid (Bouin’s solution) 3. It should be able to dehydrate even fatty tissues
3. Alcoholic iodine- used to remove excessive 4. It should not harden tissues excessively
mercuric fixatives 5. It should not remove stains
6. It should not be toxic to the body
7. It should not be a fire hazard

Commonly used:
- Alcohol (most common)
- Acetone
- Dioxane 4 – cellosolve
- Triethyl phosphate
- Tetrahydrofuran
ALCOHOL 37C temp – hastens dehydration Anhydrous
- Routine dehydration of tissues copper sulphate
- Mixes with water and other organic solvents – accelerates dehydration
- blue discoloration will indicate full saturation
Methyl Alcohol –blood and tissue films; smear preparation
Butyl Alcohol – plant and animal micro-techniques ACETONE
- Urgent biopsies
- More miscible with epoxy and other resins & CHAPTER 6
highly flammable CLEARING – de-alcoholization
- Small pieces of tissues due to extreme volatility - Alcohol or dehydrating agent is removed from the tissue
and inflammability and replaced with a substance that will dissolve the wax
with which the tissue is to be impregnated.
DIOXANE
- Less tissue shrinkage as compared to alcohol dehydration CHARACTERISTICS OF A GOOD CLEARING AGENT
- Its vapour produces a cumulative and highly toxic 1. Should be miscible with alcohol.
action in man 2. Should be miscible with, and easily removed by
melted paraffin wax
I. Gaupner’s Method 3. Should not produce excessive shrinkage
1st Pure dioxane solution 1 hr 4. Should not dissolve out aniline dyes
2nd Pure dioxane solution 1 hr 5. It should not evaporate quickly in a water bath.
3rd Pure dioxane solution 2 6. Should make tissues transparent.
hrs 1st Paraffin wax 15 minutes
2nd Paraffin wax 45 minutes 1. Xylene ( most common)
3rd Paraffin wax 2 hrs 2. Toluene
3. Benzene
II. Weiseberger’s Method 4. Chloroform
- the tissue is wrapped in a gauze bag and suspended in 5. Cedarwood Oil
a bottle containing dioxane and a little anhydrous 6. Aniline Oil
calcium oxide. 7. Clove Oil
8. Carbon tetrachloride
CELLOSOLVE
- can be stored in for months without distortion. XYLENE (XYLOL)
- Routine histologic processing
TRIETHYL PHOSPHATE - Urgent biopsies which it clears within 15-30 minutes
- removes water very readily and produces very - Becomes milky when an incompletely dehydrated tissue
light distortion and hardening of tissues is immersed in it.

TETRAHYDROFURAN TOLUENE
- dehydrates and clears tissue - Substitute for xylene or benzene
- THF is toxic if ingested or inhaled. Vapors cause - Miscible with both absolute alcohol and paraffin
nausea, dizziness, headache and anesthesia. - Much more expensive than xylene
- May cause conjunctival irritation
BENZENE
Phenol 4% + 95% Ethanol – acts as softener for hard - Urgent biopsies and routing purposes
tissues such as tendons , nails or dense fibrous tissue. - Excessive exposure to benzene may be extremely toxic
to man and may become carcinogenic or it may damage
the bone marrow resulting in aplastic anemia.

CHLOROFORM
- Slower action than xylene
- Recommended for tough tissues, for nervous tissues,
lymph nodes and embryos because it causes
minimum shrinkage and hardening of tissues

CEDARWOOD OIL
- Used to clear both paraffin and celloidin sections
- Recommended for CNS tissues and cytological
studies, particularly smooth muscles and skin.
- Becomes milky upon prolonged storage and should not
be filtered before use.
ANILINE OIL –clearing embryos, insects and very without excessive tissue shrinkage
delicate specimens due to its ability to clear 70% alcohol
 CLOVE OIL – Tissues become brittle, aniline dyes re not BY AUTOMATIC PROCESSING
removed and celloidin is dissolved. - Use of automatic tissue processing machine which
fixes, dehydrates, clears and infiltrates tissues.
CARBON TETRACHLORIDE – Similar to chloroform - Only 2-3 changes of wax are required to remove
except it’s a lot cheaper. the clearing agent
Agitation & Heat – hastens automatic process
METHYL BENZOATE AND METHYL SALICYLATE Ex: Elliott Bench-Type Processor
- used in double embedding techniques
BY VACUUM EMBEDDING
- Principle is the negative atmospheric pressure
CHAPTER 7 hastens the tissue processing
IMPREGNATION AND EMBEDDING - Removal of air bubbles and clearing agent from
IMPREGNATION - process whereby the clearing agent is the tissue block thereby promoting a more rapid
completely removed from the tissue wax penetration of tissue.
EMBEDDING – process by which impregnated tissue is - gives the fastest result
placed into a precisely arranged position in a mold
containing a medium which is then allowed to solidify SUBSTITUTES FOR PARAFFIN WAX
PARAPLAST
1. Paraffin wax - Mixture of highly purified paraffin and synthetic
2. Celloidin plastic polymers
3. Gelatin - Melting point of 56-57c
4. Plastic - More elastic and resilient than paraffin wax

55-60C – above the melting point of wax Embeddol – similar to paraplast; melting point 56-58C
56C – temp of wax normally used for routine work Bioloid – recommended for embedding eyes
Tissue Mat – Product of paraffin, containing rubber, with
1. By manual processing the same property as paraplast.
2. By automatic processing
3. By vacuum embedding ESTER WAX
- Melting point 46-48C
BY MANUAL PROCESSING - Harder than paraffin wax
- 4 changes of wax are required at 15 minutes interval - 3-4 changes of wax are required to ensure
in order to insure complete removal of the clearing complete tissue impregnation
agent.
WATER SOLUBLE WAXES
Example of time schedule for manual - Melting points 38-42C or 45-56C
processing: FIXATION: 10% Buffered Formalin Carbowax
24 hours
DEHYDRATION: 70% Alcohol 6H
95% Alcohol 12H CELLOIDIN IMPREGNATION
100% Alcohol 2H - purified form of nitrocellulose soluble in many solvents,
100% Alcohol 1H with large hollow cavities which tend to collapse, for hard
100% Alcohol 1H and dense tissues such as bones and teeth for large
CLEARING: Xylene or Toluene (2x) 1H tissue sections of the whole embryo
IMPREGNATION: Paraffin Wax (4x) 15minutes - supplied in thin 2%, medium 4%, or thick 8%
EMBEDDING: Paraffin Wax 3H
2 Types:
1. Parloidoin – hard, amber, platelet-chips
2. Low Viscosity Nitrocellulose – “nitrated variety”
- it can be used in higher concentrations and
still penetrate tissues rapidly.
- usually marketed while wet with alcohol.
2 Methods: brain sections and whole organs
1. Wet Celloidin Method Process:
- Recommended for bones, teeth, alrge Fixation  placed in equal parts of ether and alcohol (12-
24hours)  thin, medium celloidin (5-7 days) thick PLASTIC (RESIN) EMBEDDING
celloidin (3-5 days)  stored in 70% alcohol - superior results for light microscopic studies
- high resolution light microscopy of tissue
2. Dry Celloidin Method - Epoxy, Polyester, Acrylic
- Recommended for processing the whole
eye sections EPOXY
- Same Principle except that of 70% Alcohol, it is - Benzoyl peroxide + epoxy (catalysts)
GILSON’S MIXTURE (equal parts of chloroform and - epoxy plastics, catalysts and accelerators
cedarwood oil) - Cyclohexene dioxide-based plastics (Spurtt) can
- Best stored in air-tight jar be obtained pure, have very low viscosity and
infiltrate fastest.
GELATIN IMPREGNATION
- Used as an embedding medium for delicate specimens Disadvantages:
and frozen tissue sections because it prevents 1. Hydrophobic
fragmentation of tough and friable tissues 2. Reduce antigenicity of embedded tissue and may
- Fixation  10% gelatin with 1% phenol (24H)  compromise the result of immunohistochemical
transferred to 20% gelatin with 1 % phenol (12H)  staining
20% gelatin with 1 % phenol  cooled to refrigerator  3. Cause sensitization if absorbed bt skin or inhalation
transferred to 10% formalin (12-24H) 4. VCD is known to be carcinogenic
-Volume of impregnating medium should be at least 25
times the volume of the tissue POLYESTER PLASTICS
- Introduced for electron microscopy

ACRYLIC PLASTICS
- made up of acrylic acid or methacrylic acid
- used for light microscopy

Ex.
1. Polyglycol methacrylate (GMA)
EMBEDDING 2. Methyl methacrylate (MMA) – undecalcified bone
- After impregnation, the tissue is placed into a mold
containing the embedding medium and this medium
is allowed to solidify.

Types of Blocking-Out Models

1. Leuckhart’s Embedding Model – L-shaped strips of
heavy brass arranged in a flat metal plate and which
can be moved to adjust the size of mold to the size of
the specimen.
2. Compound Embedding Unit – interlocking plates
resting on a flat metal base
3. Plastic Embedding Rings and Base Mold
- special stainless steel base mold fitted with
plastic- embedding ring
4. Disposable Embedding Models
-
LABORATORY - Squash Preparation (Crushing)
- Smear Preparation
1 – FRESH TISSUE EXAMINATION -Streaking
Fresh Tissue Examination – has advantage of examining -Spreading
the tissue in living state allowing protoplasmic activities to -Pull-apart
be observed. -Touch Preparation
-Frozen Section
Methods
- Teasing (Dissociation) 2- FIXATION
Fixation – first and most critical step in histotechnology. 5- INFILTRATION/IMPREGNATION
- 10% Neutral buffered formalin is mostly widely - Impregnation is the complete removal of the clearing
used fixative because it is compatible with most agent by substitution as the medium (paraffin)
stans. penetrates the tissue with the use of no less than two,
- Physical characteristics to note: color, and preferably three baths of paraffin.
consistency, texture and size of tissue to be - Infiltrating Mediums are paraffin wax, celloidin,
processed gelatin and plastic
- Size: 1 x 1 x 0.5 -Tissue into a beaker with melted paraffin wax 15minutes
 Second beaker of paraffin wax 15 min  Third beaker
3- DEHYDRATION of paraffin wax 15 min
- Dehydration is the removal of intercellular and
extracellular water from the tissue following
fixation. 6 – EMBEDDING
- Alcohol is the most commonly used dehydrating agent - Embedding is the orientation of the tissue in melted
- Acetone provides a rapid method “stat” method paraffin, which when solidified, provides firm medium
- Dioxane is a rapid dehydrating agent but its fumes for keeping intact all the parts of the tissues when
are highly toxic sections are cut.
-Cellosolve dehydrated rapidly and is not harmful to the - Leuckheart’s Embedding Model
tissues - Compound Embedding Unit
- Tetrahydrofuran possesses same properties as dioxane - Plastic Embedding Unit
-70%Ethyl alcohol 6H  90%Ethyl Alcohol 12H  - Disposable Embedding Molds
Absolute Ethyl Alcohol 2H  Absolute Ethyl Alcohol 1H  -Paper boats, Peel-away, Plastic ice trays
Absolute Ethyl Alcohol -Arrange tissue in embedding mold  Tissue is oriented at
the center of the mold  Pour melted paraffin wax in a
4- CLEARING (DE-ALCOHOLIZATION) mold and allow to solidify for 3 H
- Clearing is the removal of dehydrating agent from
the tissue MANUAL TISSUE PROCESSING
-Xylene is the most rapid clearing agent, suitable for FIXATION: 10% Buffered Formalin 24 hours
urgent biopsies DEHYDRATION: 70% Alcohol 6H
- Xylene 1H  Xylene 1 H 95% Alcohol 12H
100% Alcohol 2H
100% Alcohol 1H
100% Alcohol 1H
CLEARING: Xylene or Toluene (2x) 1H
IMPREGNATION: Paraffin Wax (4x) 15minutes
EMBEDDING: Paraffin Wax 3H