SU-ICLS HISTOPATHOLOGY SECTION

TISSUE PREPARATION

Procedure:
1. Use complete and proper PPE.
2. Prepare the following reagents:
10% FORMALIN 37-40% formaldehyde 100 mL
Distilled water 900 mL
80% ETHANOL 95% ethyl alcohol 84.2 mL
Distilled water 15.8 mL
70% ETHANOL 95% ethyl alcohol 73.7 mL
Distilled water 26.3 mL
1% ACID ALCOHOL 70% ethyl alcohol 100 mL
Concentrated HCl 1 mL
AMMONIA WATER Distilled water 100 mL
Strong ammonia 1 mL
HARRIS Hematoxylin powder 1 gram
HEMATOXYLIN Absolute ethyl alcohol 10 mL
Ammonium/potassium 20 grams
alum
Distilled water 190 mL
Mercuric oxide 0.5 gram
Glacial acetic acid 10 mL
EOSIN (STOCK Eosin Y 1 gram
ALCOHOLIC Distilled water 20 mL
SOLUTION) 95% ethyl alcohol 80 mL
MAYER’S EGG Egg white 50 mL
ALBUMIN Glycerin 50 mL
ALCOHOLIC 37-40% formaldehyde 100 mL
FORMALIN 80% ethyl alcohol 900 mL
NOTE:
a. For Harris Hematoxylin:
1. Dissolve hematoxylin powder in absolute ethyl alcohol with gentle heating.
2. Dissolve ammonium/potassium alum in distilled water on a large (500 mL capacity)
boiling flask or beaker.
3. Add hematoxylin solution and boil.
4. Add mercuric oxide and plunge immediately into cold water for rapid cooling. The violent
liberation of oxygen will cause the solution to explode from a narrow-mouthed flask hence the
need for a large beaker.
5. The solution is then filtered and transferred into a well-stoppered bottle and remains
stable for a long time. The solution should assume a dark purple color upon addition of mercuric
oxide. The addition of 4% glacial acetic acid will give a more precise nuclear staining.
b. For Eosin
1. Dissolve Eosin Y in distilled water by gentle heating.
2. Cool and add 95% ethyl alcohol. For use: 1 part of the stock solution is usually diluted
with 3 parts of 80% ethyl alcohol. Addition of 0.5 mL glacial acetic acid for every 100 mL of stain
will usually give a deeper red stain to the tissue.
c. For Mayer’s Egg Albumin
1. Mix well then filter. Add thymol crystals
SU-ICLS HISTOPATHOLOGY SECTION

Variation in the methods of tissue preparation exists because of some structural and chemical
components of the cell that an examiner wanted to sought, the nature and amount of the tissue
to be evaluated and the need for an immediate examination of a tissue. A fresh tissue
preparation is favorable when enzymes, lipids, motion, phagocytosis, pinocytosis and when an
immediate need is desirable. But a fixed and preserved tissues are frequently used because the
chemicals in the tissue process arrests decay and the tissue structures can be studied for prolong
period of time compared to fresh ones.
Methods of fresh tissue examination include the following: teasing or dissociation, squash
preparation or crushing, frozen preparation and smear preparation. The smear preparation can
also be prepared by streaking, spreading, pull-apart, and touch preparation or impression smear.
For frozen section, it is normally utilized when rapid diagnosis of the tissue is required. A 10-15
microns in thickness is cut in a microtome with CO2 or in a cryostat machine.

For a permanently preserved, stained and well-mounted tissue on slides with coverslip, it
provides an ample time of studying tissue and permanent keeping for future reference. Preserved
tissues are done in the following order: fixation, dehydration, clearing, infiltration, or impregnation,
embedding, trimming, section-cutting, staining, mounting, and labeling.

Prior to tissue preparation, tissue accessioning is done to avoid errors in the process. The
following date should be noted:
 Type of tissue (autopsy or biopsy)
 Site of origin
 Date of extraction
 Code number (patient’s name, age, initial diagnosis)
 Specimen number (1st, 2nd, 3rd, etc.)

When done, a gross examination is done by the pathologist. He takes note of the physical
characteristics like the size, shape, weight, color or even consistency of the specimen. For solid
specimens, a cut tissue can done by either in midsagittal, frontal and transverse section following
the required thickness and length. A recommended thinness of a fresh tissue cut should be 3-5
mm or less than this.

Procedure:
1. Cut the tissue assigned from the dissected specimen.
2. Tissue should not be more than 5 mm thick except in lung edema with minimum squeezing
and handling.
3. Place the cut tissue inside the tissue cassette or tissue capsule.
4. With a string, tie the tissue capsule. At the other end of the string, write the identification
number or label.
5. Immerse in 10% BNF for fixation.
SU-ICLS HISTOPATHOLOGY SECTION

FIXATION AND DECALCIFICATION

Fixation is the process of using either physical or chemical methods of fixing living tissues
such that cells and extracellular materials must be preserved with as little alterations as possible
to its structure and chemical composition. A perfect fixation is theoretically and practically
impossible to attain. However, a skillful and experienced Medical Technologist keeps on trying to
achieve such.

PRIMARY GOAL OF FIXATION - To preserve the structure especially the lipoproteins

and carbohydrates making up the cell and its organelles.
The most common physical examination done is by heat application. Chemical fixatives
vary according to its composition and action. The use of 10% formalin is a common fixative used
in the laboratory. Other fixatives include mercuric chloride, picric acid, acetone, Bouin’s fluid, etc.

Hard specimens such as bones, cartilages, teeth, calcified lungs and atherosclerotic
vessel which contain a large amount of calcium salts cannot be easily proceed to other steps
unless the calcium or lime salts are removed. The process of removing calcium salts in those
specimens is called decalcification. This process is done after fixation and prior to infiltration
or impregnation to facilitate cutting of tissues. The tissues are thoroughly washed to remove the
fixatives to avoid undesirable chemical reactions (e.g. mercury would form a chelate with EDTA,
or the residual sodium phosphates would oppose to the action of an acid decalcifier].

Decalcification is done by using acids like nitric acid, HCl, formic acid, TCA, sulphorous
acid, chromic acid, citric acid, chelating agents like EDTA, ion exchange resins and electrical
ionization (electrophoresis).

Procedure:
1. Immerse desired size of tissue specimen into 10% BNF for 24 hours.
2. Transfer to container labeled alcoholic formalin 1 and alcoholic formalin 2 for 1 hour each.

DEHYDRATION

The presence of water intracellularly and extracellularly will hinder the tissue processing
unsuccessful this shall be removed to allow the embedding medium to penetrate inside the tissue.
A good dehydrating agent must possess the following characteristics: dehydrate the tissue rapidly
without producing considerable shrinkage or distortion, should not evaporate very fast, can
dehydrate even fatty tissues, doesn’t harden tissue excessively, does not remove stains, not toxic
to the body and not a fire hazard.

Commonly used dehydration agents are: alcohol (most common), acetone, dioxane,
cellosolve, triethyl phosphate and tetrahydrofuran. Ethanol is the best dehydrating agent
because it is fast acting, it mixes with water and penetrates tissue easily. It is not poisonous and
not very expensive, are used for routine purposes. Acetone is fast-acting thus used in urgent
biopsies. Dioxane (diethylene dioxide) is an excellent dehydrating and clearing agent readily
miscible n water, melted paraffin, alcohol and xylene. It produces less tissue shrinkage as
compared to alcohol dehydration. It is expensive, dangerous and highly toxic action in man.
Cellosolve dehydrates rapidly and is not harmful to the tissues. Triethyl phosphate removes
water very readily and produces very little distortion and hardening of tissue. It is soluble in
alcohol, water, ether, benzene, chloroform, acetone and xylene. It is used to dehydrate sections
and smears following certain stains and produces minimum shrinkage. Tetrahydrofuran both
SU-ICLS HISTOPATHOLOGY SECTION

dehydrates and clears tissues since it is miscible in water and paraffin. It can dissolve many
substances including fats and is miscible in lower alcohol, ether, chloroform, acetone, benzene
and xylene. It may be used for demixing, clearing and dehydrating paraffin sections before and
after staining.

Procedure:
1. Prepare enough amount of ascending solutions of alcohol into four, wide mouth containers
with tight covers. Label containers Alcohol I, II, III and IV.
2. Immerse previously fixed tissue in 4 ascending changes of alcohol for 1 hour each.
3. Presence of turbidity in the process indicates the removal of water from the tissue

CLEARING

Clearing is also known as dealcoholization. It is the process of removing alcohol or any

dehydrating agent from the tissues, replacing it with the one that will be miscible with the
embedding medium (e.g. paraffin wax) to be used. Presence of clearing agent increases tissue
transparency and improves their refractive indices for better optical differentiation of cells. The
most common, excellent and true clearing agent is xylene. Other clearing agents are toluene,
benzene, chloroform, cedarwood oil, aniline oil, clove oil, carbon tetrachloride, petroleum ether,
histoclear, oil of wintergreen, oil of bergamot, oil of origanum, dioxane, terpineol, carbon disulfide
and aviation gasoline. Benzene is hazardous, causes aplastic anemia and cancer and is the most
toxic. Chloroform is excellent for nervous tissues, brain, lymph nodes, and embryos. The most
recently introduced clearing agent is histoclear. Toluene is a substitute for xylene or benzene. It
is miscible in dehydrating agent and embedding medium.

A good clearing agent must possess the following characteristics: must be miscible with alcohol
and paraffin, should not produce excessive tissue shrinkage and hardening, should not dissolve
out aniline dyes, should not evaporate quickly in a water bath and should make tissues
transparent.

Procedure:
1. Prepare enough amount of 2 changes of xylene on big, wide-mouthed containers with
tight-fitting cover. Proper labeling should be made.
2. Transfer immediately dehydrated tissues on Xylene 1 and immerse for 1 hour.
3. Transfer to Xylene II and immerse for another 1 hour. This makes a total of 2 hours. Do
not immerse tissue for more than the required time otherwise the tissue will get brittle and hard.

INFILTRATION AND EMBEDDING

Infiltration is also known as impregnation. It is the process whereby the clearing agent is
completely removed from the tissue and replaced it by a medium that fill up the tissue cavities to
give a firm consistency to the specimen to facilitate support for easy cutting producing a thin,
transparent tissue. The routine and common impregnating medium used in most of the
laboratories is paraffin wax. The melting point of the paraffin wax is between 56-58 degrees
centigrade. But if the temperature of the environment is 15-18 degrees centigrade, the melting
point of the paraffin wax is between 50-54 degrees centigrade. It must be noted that the volume
of impregnating medium should be at least 25 times the volume of the tissue.
SU-ICLS HISTOPATHOLOGY SECTION

Embedding is also known as casting or blocking. It is the process by which the

impregnated/infiltrated tissue is placed into a precise position in a molder containing medium
(paraffin wax) is allowed to solidify. This gives an external support to the impregnated tissue.
Other embedding medium are:
1. Celloidin – for extradensely cellular tissues such as bones, cartilages, brain, embryos, and
specimens with large, hollow cavities (e.g. eyes).
2. Gelatin – used when dehydration is to be avoided and when tissues are subjected for
enzymatic studies, for delicate specimens.
3. Paraffin wax substitute
a. Paraplast – is a mixture of highly purified paraffin and synthetic plastic polymers,
has a melting point of 56-57 degrees centigrade.
b. Embeddol – synthetic wax substitute, similar to paraplast, has a melting point of
56-58 degrees centigrade.
c. Bioloid – synthetic wax, recommended for embedding eyes
d. Tissue Mat – a product of paraffin, containing rubber, similar properties with
paraplast.
e. Ester Wax – has a lower melting point of 46-48 degrees but is harder than paraffin.
f. Carbowax – polyethylene glycols with melting points of 38-42 degrees centigrade
or 45-56 degrees centigrade.

Molders used in the embedding procedure are:

1. Leuckhart’s embedding – consist of two L-shaped strips of heavy brass or metal arranged
on a flat metal plate, recommended for routine use.
2. Compound embedding unit – made of series of interlocking plates resting on a flat metal
base, forming several compartments.
3. Plastic embedding rings and base mold – consists of a special stainless steel base mold
fitted with a plastic embedding ring which later serves as the block holder during cutting.
4. Tissue Tek – a machine equipped with a warm plate to manage the impregnated specimen
and a cold plate at a 5 degrees centigrade for rapid solidification of the block.
5. Disposable embedding molds
a. Peel-away – disposable thin plastic embedding molds, available in 3 different sizes
are simply peeled off one at a time as soon as the wax has solidified.
b. Plastic Ice Trays – those used in ordinary refrigerators, recommended for busy
routine laboratories.
c. Paper Boats – useful for embedding celloidin and paraffin wax blocks

Procedure:
1. Label 3 tin cups as Paraffin 1, Paraffin 2, and Paraffin 3 (for embedding purposes).
2. Fill in large tin cups about ¾ full with paraffin wax.
3. Place tin cups in boiling water until the wax melts at its point. Make sure that the
maintained temperature of the melted wax is between 56-58 degrees centigrade.
4. Transfer immediately cleared tissue cassette in the following order:
a. Paraffin 1 = 1 hour
b. Paraffin 2 = 1 hour
5. Remove tissues from the cassette and orient at the bottom of the appropriate molders.
6. Add melted paraffin wax as the embedding medium and label properly. Allow to solidify
and cool rapidly by placing it inside the refrigerator at -5 degrees centigrade or immerse
in cold water for complete solidifaction.
SU-ICLS HISTOPATHOLOGY SECTION

TRIMMING AND TISSUE SECTIONING

A solidified tissue block is removed from the molder and using a sharpened knife, excess
paraffin wax is perfectly shed off from both sides and on surface of the block showing off a bit of
the exposed tissue. The result is a four-sided rectangular prism, this step is called trimming.
Make sure that only thin slices of excess wax are taken out to prevent the block from cracking.
The moment the trimmed tissue block is mounted on a microtome and is manipulated for cutting,
this step is called sectioning. Sectioning is a process whereby tissues are cut into thin slices
measured in micra or millimicra using equipment called microtome. Depending on the quality,
type of embedding medium used and necessity of results to be obtained, a specific microtome is
used.

PARTS OF THE MICROTOME

 Flywheel – is used for manual manipulation; has a lock
 Pawl – controls and regulate the number of rotations
 Ratchet feed wheel – determines the number of rotations made by the wheel
 Micrometer – determines the thickness of the section cut
 Knife holder – hold the knife in place
 Chuck – holds the tissue block
 Base – supports the entire structure

KINDS OF MICROTOME
 Rotary Microtome – for cutting paraffin-embedded sections
 Sliding Microtome – for cutting celloidin-embedded sections
 Freezing Microtome – for cutting unembedded frozen sections for the demonstration of
lipids, fats, and enzymes
 Ultrathin Microtome –cutting sections for electron microscopy
 Rocking Microtome – cutting serial sections of large blocks of paraffin-embedded tissues

PRINCIPLE OF MICROTOME
A spring-balanced teeth or pawl is brought into contact with and turns a ratchet feed wheel
connected to a micrometer screw, which is in turn rotated, moving the tissue block at a
predetermined distance towards the knife for cutting sections at uniform thickness.
Procedure:
1. Remove excess paraffin wax using sharpened knife forming into four-sided rectangular
prism. Cut must be parallel on four sides.
2. Place tissue on knife holder or chuck. Set micrometer to 5 um when ready to cut tissue.
3. Cut gently.
4. Place cut tissue on a heated water bath maintaining a temperature of 45-50 degrees
centigrade or 6-10 degrees below the melting point of the wax used for embedding the
tissue.
5. A flattened chosen tissue is vertically picked up using a clean slide with adhesive by
immersing into the water.
6. Place slide into paraffin oven for drying. *The use of 20% alcohol or distilled water are
alternatives to flatten out ribboned-cut tissue. Done by adding them into slide with cut
tissue.

STAINING
SU-ICLS HISTOPATHOLOGY SECTION

Staining of tissue sections enable the histotechnologist appreciates and differentiates cell
constituents according to the type of stains they have affinity with. These staining reactions are
made possible because of the following mechanism such as physical and chemical. The
physical mechanism includes: adsorption, capillary osmosis, solubility and absorption of stains or
dyes by the tissue or cell concerned. On the chemical basis, there are certain parts of the cells or
tissue which are acidic or basic in nature. An acid cell part (nucleus) has an affinity with the basic
dye while a basic cell part (cytoplasm) has the affinity with an acid dye.

There are a number of dyes employed to stain tissues which is classified as natural dyes and
synthetic dyes. Example of natural dyes are cochineal dyes, logwood dyes and vegetable extract
dyes. Synthetic or artificial dyes are aniline and coal tar dyes. The most common dye used in the
laboratory is Hematoxylin and Eosin staining when using paraffin wax as the embedding medium.
Hematoxylin and eosin are utilized for microanatomical studies of tissues using a regressive
staining. In regressive staining, the tissue is overstained to obliterate the cellular details, and the
excess stain is removed or decolorized from unwanted parts of the tissue by the use of acid-
alcohol.

PRINCIPLE OF STAINING
It is based on the chemical reaction between the tissue and the dye in order to produce
affinities between them for better color contrast, optical differentiation, for microscopic
visualization and to improve aesthetic value.

Stains based on reaction (pH):

 Basic stains have a pH of above 7.0, also known as your primary dye, are basophilic and
has a violet or dark blue in color. Example are: hematoxylin, thionine, crystal violet, gentian
violet, methyl violet, methylene blue, toluidine blue and lugol’s fast blue.
 Acidic or cytoplasmic stain have a pH below 7.0 and is also called secondary or counter
stain and is either light pink, red or orange in color. Examples are: eosin, carmine, safranin,
orcein carbol fuchsin, congo red and methyl red.
 Natural or amphoteric stain have a pH equal to 7.0 where the acid and the base are
almost equal, and is pale blue in color. Examples are: wrights, giemsa, leishmans,
Wolbach-mc namara and supravital dyes.

Staining Methods in Cytology

Papanicolaou staining method is the routine staining procedure used in cytopathology
laboratory. It is a polychrome staining reaction that results in well stained nuclear chromatin,
differential cytoplasmic counterstaining and cytoplasmic transparency. Cytoplasmic stains are
OG-6 and EA-36. Both are synthetic stains and OG-6 is a monochrome stain while EA-36 is a
polychrome stain. Formalin-fixed cytology preparations must be stained with either H&E or
Papanicolaou stain.
To date, Papanicolaou (Pap) smear is considered to be the staining method of choice for
exfoliative cytology. This staining technique, known as the Pap stain, is still the gold standard
today. Sputum or urine specimens containing squamous epithelial cells also show excellent
results when stained according to the Papanicolaou technique.

Procedure: Hematoxylin and Eosin Staining

Xylene I 2 minutes
Xylene II 2 minutes
Xylene III 2 minutes
SU-ICLS HISTOPATHOLOGY SECTION

95% ethyl alcohol 10 dips

80% ethyl alcohol 10 dips
70% ethyl alcohol 10 dips
Distilled water 10 dips
Harries Hematoxylin 2 minutes
Tap water 10 dips/overflow
Acid-alcohol 2 dips
Tap water 10 dips
Ammonia-water 5 minutes/until blue
Tap water 10 dips
Eosin 1 minute
70% ethyl alcohol 10 dips
80% ethyl alcohol 10 dips
95% ethyl alcohol 10 dips
Xylene I 10 dips
Xylene II 10 dips
Xylene III 10 dips/until clear

Then mount your stained tissues using mounting medium

Procedure: Papanicolau Staining

Distilled water 10 dips

Harris hematoxylin 1 minute
Tap water 3 sets;10 dips each
Polychromatic stain 1 minute
80 % ethyl alcohol 10 dips
2-propanol 10 dips
2-propanol 10 dips
Xylene 10 dips
Xylene 1 minute

MOUNTING, LABELLING, AND FILING

Mounting is a step of putting a syrupy fluid to sectioned tissue under the coverslip to protect
the section from physical injury, bleaching or deterioration due to oxidation This assumes
permanency of the tissue, improve refractive index and for better microscopic visualization.
Application of mountant is done by placing a clean coverslip on a sheet of filter paper, the
sectioned tissue, while still wet with xylene, is placed with few drops of mountant and the slide is
quickly inverted and lowered to the coverslip. A slight pressure is applied till the mountant flows
evenly to the edge of the coverslip and the slide is quickly turned over. There are two types of
mountant: aqueous or temporary mountant and resinous or permanent mountant. Examples
of aqueous mounting media are farrants, kaissers, apathy’s, karo corn syrup, glycerin jelly, water
and brun’s fluid. Natural resinous mounting media are Canada balsam, gum gammar, DPX,
XAM, permount and clarite. Synthetic resinous mounting media are harfmanni, eukitt, diatexx,
protexx, and clearmount.
SU-ICLS HISTOPATHOLOGY SECTION

CHARACTERISTICS OF A GOOD MOUNTING MEDIA

1. refractive index as near as possible to the glass slide which is 1.518 to avoid distortion of
image
2. should not dry quickly
3. should not dissolve out or fade tissue sections
4. will not cause shrinkage and tissue distortion
5. it should set hard thereby producing permanent mounting of sections.

Slides for mounting should be properly labeled with case number for identification purposes
and avoid accidents like wiping the wrong side of the slide containing the tissue section. Ways of
labeling may be done by using permanent and temporary label materials. Special glass pencil
and gum label can serve permanently while use of colorless nail polish gives temporary purpose
only.
Ringing media added for more advantage to the preparation. A ringing is used to seal the
margins of the coverslips so that evaporation and escapage of the mountant are prevented. A
histotechnologist may use kronig cement or durofix.

Filing of the prepared tissue slides must be systematic and chronologically arranged for future
reference, in research work and scientific study.

Procedure:
1. Clean the slides after staining.
2. Place a drop of mounting medium on the slide.
3. Place a coverslip and allow the mounting medium to spread evenly.
4. Apply a light pressure to remove excess mountant as well as bubbles, if any.
5. Excess mountant is removed by using xylene and water.
6. After the mountant has dried, clean the prepared slide.
7. Write the name of the tissue or specimen as well as its code number on a gum label.
8. Let it stick on the slide for identification.
9. Place your prepared slide in a filing case.
SU-ICLS HISTOPATHOLOGY SECTION

ROUTINE TISSUE PROCESSING

PROCEDURE:
A. Dissection and Numbering of Tissues
1. Cut the tissue assigned from the dissected specimen.
2. Tissues should not be more than 5mm thick except in lung edema (in which case
tissue slices may be 1-2cm thick) with minimum squeezing and handling.
3. Place the tissue inside the tissue cassette/capsules.
4. With a string, tie the tissue capsule. At the other end of the string, write the
identification number/label.
5. Immerse in 10% formalin for fixation.

B. Processing of Tissues for Histotechnology

10% Buffered Neutral Formalin
Formalin I 7:00AM
Formalin II 9:00AM
95% ethanol I 10:00AM
95% ethanol II 11:00AM
Absolute alcohol I 12:00NN
Absolute alcohol II 1:00PM
Xylene I 2:00PM
Xylene II 3:00PM
Paraffin I 5:00PM
Paraffin II 6:00PM
Embedding 7:00PM

C. Trimming and Sectioning

1. With a knife, trim the excess waxes from all sides of the block so that the block forms
a four-sided prism (rectangular), opposite sides being parallel to expose the tissue
surface in preparation for actual cutting.
2. At least 2mm of wax should surround the block.
3. The block is allowed to harden for cutting proper by facing it down in ice cubes or cold
water or places in refrigerator for 5-10 minutes.
4. The block is then placed in the microtome for cutting.
5. The microtome blade is placed into the blade holder in its proper position and angle
(14 degrees).
6. Set the micrometer (4-6u for rotary microtome) to cut the tissue block into desired
thickness.
7. Rotate the flywheel to determine the sections and form ribbons.
8. If the ribbon crumbles, apply ice to harden the tissue block.
9. Transfer ribbons from the microtome to the flotation bath. Flotation bath should be
maintained at 45-50 degrees.

D. Floating-out
1. The ribbons are gently placed on the floating-out bath to flatten the tissue ribbons.
2. With the use of a needle, separate sections for easy fishing-out.
SU-ICLS HISTOPATHOLOGY SECTION

E. Fishing-out
1. Place a drop of Mayer’s egg albumin on the slide and spread evenly.
2. Fish out the tissue sections from the floating-out bath to the slide, taking care not to
crumble it.
3. Wipe off the melted paraffin wax with a clean cloth.

F. Staining
Xylene I 2 minutes
Xylene II 2 minutes
Xylene III 2 minutes
95% ethyl alcohol 10 dips
80% ethyl alcohol 10 dips
70% ethyl alcohol 10 dips
Distilled water 10 dips
Harries Hematoxylin 2 minutes
Tap water 10 dips/overflow
Acid-alcohol 2 dips
Tap water 10 dips
Ammonia-water 5 minutes/until blue
Tap water 10 dips
Eosin 1 minute
70% ethyl alcohol 10 dips
80% ethyl alcohol 10 dips
95% ethyl alcohol 10 dips
Xylene I 10 dips
Xylene II 10 dips
Xylene III 10 dips/until clear

G. Mounting/Coverslipping
1. Clean the slides after staining.
2. Place a drop of mounting medium on the slide.
3. Place a coverslip and allow the mounting medium to spread evenly.
4. Apply a light pressure to remove excess mountant as well as bubbles, if any.
5. After the mountant has dried, clean the prepared slide.

H. Labeling and Filing

1. Write the name of the tissue or specimen as well as its code number on a gum label.
2. Let it stick on the slide for identification.
3. Place your prepared slides in a filing case.