MLS 410 LEC – HISTOPATHOLOGIC AND

CYTOLOGIC TECHNIQUES
INFILTRATION, EMBEDDING & SECTIONING
2ND SEMESTER | MIDTERM | PROF. NIEL JOHN MARAVILLA

○ Incomplete Impregnation → Air Holes in tissue

OUTLINE sections.
I. Impregnation & Embedding
■ This will manifest during sectioning.
A. Define Impregnation and Embedding.
Mahihirapan kayo magcut dahil sa incomplete
B. Discuss Different Factors Affecting Impregnation
and Embedding. impregnation (or butas-butas).
C. Identify Impregnation and Embedding Agents and 3 TYPES OF GENERAL TISSUE IMPREGNATION
their Actions to Tissues. 1. PARAFFIN WAX IMPREGNATION
D. Observe Proper Orientation of Tissues to Tissue 2. CELLOIDIN IMPREGNATION
Blocks. 3. GELATIN IMPREGNATION
II. Sectioning 1. PARAFFIN WAX

IMPREGNATION & EMBEDDING

■ These two procedures are often grouped
together because routinely they use the same media.
Although some procedures require different media for
impregnation and different media for embedding.
■ Yet ROUTINELY they used the same
infiltrating medium.

● Requires 2 or more changes of melted paraffin

wax.
○ Melting Point: 45°C, 52°C, 56°C (routine), 58°C
@RT (20-24°C)
■ Impregnation and Embedding are usually ● Never overheat (> 60°C) - causes brittleness,
done after clearing. shrinkage, hardening; destruction of lymph tissue.
IMPREGNATION / INFILTRATION ● Paraffin oven must be maintained 2-5°C above the
MP of wax
■ If your paraffin wax has a melting point of
56°C your oven must be around 58-61℃.
● Used pure (without additives)
○ Wax must be filtered first (done manually) using
coarse filters such as Green's nO. 904 in a wax oven at 2°C
higher than MP of wax.
● WHAT IS IMPREGNATION? ○ Reusable only once, but heat it first @100-105°C
○ Process that removes the clearing agent (remove water)
■ Since the process before is clearing, the ■ In an ideal laboratory setting we don’t
tissue now is filled with a clearing agent. So, we need reuse paraffin wax because if we reuse paraffin wax
to remove the clearing agent for you to be able to fill it magkakaroon ng carry over ng tissue sample.
with the infiltrating media. ■ Yung mga tissue cassettes natin may mga
■ One of the qualities of a clearing agent is it holes diba, what if yung tissue sample na negative for
should be miscible with the infiltrating media para mas cancer kay ma mix with ng tissue sample na positive
mabilis makapasok ang infiltrating media at palitan for cancer. Contamination will lead to misdiagnosis
ang clearing agent. that’s why hindi sya nirereuse.
○ Fills the tissue cavities
■ The infiltrating media will enter the tissue ADVANTAGES:
and will solidify to create a support medium for the ● Rapid (24 hours)
tissue. So. that our tissue will have a structure ● Maybe cut with ease without undue distortion.
integrity for it to be able to permit tissue sectioning. ● Many staining procedures are permitted.
○ Permeates tissue with a support medium

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INFILTRATION, EMBEDDING & SECTIONING

■ If ang staining hindi mag incorporate well

with your paraffin wax at hindi talaga sila magmatch SUBSTITUTE FOR PARAFFIN WAX
hindi ka makaproduce ng good quality slides. If your ● PARAPLAST MP: 56-57℃
staining does not permit paraffin wax better not use ■ Mixture of pure paraffin and synthetic
paraffin wax. plastic polymer (Dimethyl sulfoxide); more elastic and
■ But ROUTINELY most staining procedures resilient.
permits paraffin wax. ● EMBEDDOL - less brittle, and less compressible
(MP: 56-57℃
DISADVANTAGES: ● BIOLOID - semisynthetic; for embedding of eyes
● Prolonged impregnation will cause excessive ● TISSUE MAT - has rubber
shrinkage. ● ESTER WAX MP: 46-48℃ (pinakamababang
● Not recommended to fatty tissues melting point)
■ You use the type of wax depending on the ■ Harder than paraffin thus used with
type of specimen, type of staining procedure, and sliding/sledge-type microtome
type of outcome you wanted to achieve. ■ Water insoluble but soluble in 96%
● Overheated paraffin makes the specimen brittle. ethanol, thus prior clearing is not needed.
- Hindi mo na kailangan mag gamit
THREE WAYS OF PARAFFIN WAX IMPREGNATION ng clearing agent kapag yung wax
1. BY MANUAL PROCESSING itself is be able to remove the
alcohol just like this ester wax.
- THIS IS NOT A ROUTINE
PROCEDURE..
■ But Cellosolve, and xylene may be used if
indicated.
- If indicated by the pathologist.
● WATER SOLUBLE WAXES (Polyethylene glycol)
MP: 38-42°C or 45-56°C
■ The reason why Ester wax ang
pinakamababa ang melting point at hindi ang water
soluble wax is because meron syang two types of
■ The image shows the manual processing. melting points.
2. BY AUTOMATIC PROCESSING (AUTOTECHNICON E.g. CARBOWAX - most common
& ELLIOT BENCH-TYPE) ■ No need for dehydration and clearing
■ Thus sections are difficult to float out and
mount.
[Remedy: add soap to water, or 10% G 900 in
water]
■ Neutral fats and lipids can be
demonstrated.
- If fats and lipids are the specimen
we don’t use routine procedures
■ Example of Elliot-bench type machine.
since ayaw natin magmeet yung
This machine is heavy, around 2 kgs if walang laman
xylene and fats.
mas mabigat kung meron.
■ For enzymes histochemistry
3. BY VACUUM EMBEDDING

2. CELLOIDIN IMPREGNATION

■ Utilizing vacuum, para mas mabilis mag

open ang mga gaps, nooks and crevices ng tissues
para makapasok ng husto ang wax. Syempre if
makapasok sila ng husto there will be no air holes na
mangyayari.

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INFILTRATION, EMBEDDING & SECTIONING

(Image analysis: These are parts of the brain na gi cut, and

submerged in 12% celloidin. Ang ginagamit na mold ky
wheaton dish).
● AKA COLLOIDIN
○ Purified form of nitrocellulose/gun cotton
○ Specimens with large and hollow cavities which
tend to collapse; hard and dense tissues; neurologic
tissues.
■ used for dako na mga specimens na
daghan ug mga gaps such as brain.
○ Concentration: in 2%, 4%, 8%, dissolved in equal
parts of ether and alcohol.
■ Other details

ADVANTAGES:
3. NITROCELLULOSE/ LOW VISCOSITY
● Does not require heat; less shrinkage
NITROCELLULOSE (gun cotton)
● Cutting of thicker tissues
■ Has lower viscosity, thus can be used in
● Recommended for neurological tissues (BRAIN)
high concentrations, and rapid tissue penetration.
■ ADV: harder tissue blocks, thus thinner
DISADVANTAGES:
sections are possible
● Very slow (days or week)
■ DAVD: Explosive when dry due to
● Thin sections are difficult to cut
nitrates
● Very volatile
■ Plasticizer (e.g oleum ricini or castor oil) is
needed to prevent tissue cracking in
METHODS OF CELLOIDIN INFILTRATION
chrome-mordanted tissue.
1. WET - For bones, brain, teeth
- Chrome-mordanted tissues is sa
■ STORE TISSUE BLOCK IN 70%-80%
staining
ALCOHOL
3. GELATIN IMPREGNATION

● Rarely used
○ For histochemical, enzyme studies, and frozen
sections (fresh tissues)
2. DRY - For whole eye sections
ADV: Water soluble (no dehydration and clearing needed)
■ STORE TISSUE BLOCK IN GILSON’S
(since the tissue is fresh)
MIXTURE (CHLOROFORM AND CEDARWOOD
DADV: May decay
OIL)
○ TSE must be <2-3 mm thick
○ 1% Phenol must be added to prevent molds.

■ For washing out fixatives it’s not a total

wash out but only an initial wash out since the tissue
is fresh. (pertains to the procedure shown in the
photo)
EMBEDDING
● WHAT IS EMBEDDING?

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INFILTRATION, EMBEDDING & SECTIONING

○ AKA Casting, Blocking, Molding PROCEDURE:

○ Placing the impregnated tissue into a mold with 1. Tissue taken out
embedding media, and then allowing the media to solidify. 2. Molten paraffin poured on mold
■ Embedding allows proper orientation of the 3. Tissue embedded in paraffin (observe proper
tissues. orientation of tissue in this step)
○ Orientation: Arrangement of the tissue in a precise 4. Tissue is pressed on mold
position in the mold during embedding. 5. The mold is covered with peripheral plastic ring
■ STORE TISSUE BLOCK IN GILSON’S 6. Unique number is put in the plastic ring
MIXTURE (CHLOROFORM AND CEDARWOOD 7. The molds are put on the cold plate
○ Surface to be cut should be parallel to bottom of - Prolonging in a cold plate may result in
the mold cracks.
■ Kung ano gusto mo ipakita sa slide it 8. Then remove the tissue
should be parallel to the bottom of the mold. Usually - Malalaman mo na okay na sya tanggalin pag
called the BLOCK FACE. madali na lang syang tanggalin. If may
○ molds should bear the accession number resistance hindi pa siya pwede tanggalin.
■ Sa parang triangle na part nilalagay ang ORIENTATION
accession number and then for back up pwede ka ● Arrangement of the tissue in a precise position in
magagay sa likod. the mold during embedding.
■ Majority ng tissues natin ay oriented kung
saan feel mo na block face niya. Used in routine.

SPECIFIC ORIENTATION:
Tubular tissue: all layers in transverse section
Skin: all layers should come
Endometrial curetting: Keep in center
Long tissue: keep diagonally
Intestine: all layers should come
Membrane: Swiss roll
PROCEDURE:
● Put tissue with label on a mold
● Immerse them in melted paraffin at 5-10℃ higher
than MP of wax (refers to paraffin oven)
● Rapidly cool in a refrigerator at -5℃, or in cold
water.

TYPE OF MOLDS
1. Leuckhart’s Embedding Mold
○ L-Shaped Metal Plate
○ An L-shaped metal plate joined together to
form a tissue block

■ This type of embedding center is called a

tissue trek system.
■ May wax dispenser sya, tissue warmers,
hot plate, forceps warmer, and cold plate.
■ The filter is already embedded in the
machine.

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2. Compound Embedding Unit ● Most widely applied, but carcinogenic (having the
○ Usually, pang maramihan na embedding. potential to cause cancer) due to vinylcyclohexene
○ Isa ka embedding unit can hold 30 or more dioxide (VCD) components.
na tissue samples. ● Types:
○ Not used routinely. ○ Bisphenol A (Araldite) - Slow
○ Glycerol (Epon) - Low viscosity
○ Cyclohexene Dioxide (Spurr) - Very low
viscosity; fastest
POLYESTER
● For electron microscopy; seldomly used

ACRYLIC PLASTIC
● For high-resolution light microscopy
● Example: Polyglycol methacrylate (GMA), methyl
methacrylate (MMA)
3. Plastic Embedding Rings and Base Mold ● Benzoyl peroxide
○ Base molds can be metallic ○ A catalyst; forms radicals, which are a site
for polymerization
● Must be stored in dark bottles, to prevent radical
formation and premature polymerization
● Embedding media may be stained, thus use
hydrophobic MMA

TRIMMING AND MICROTOMY PROCESS

● Trimming of tissue → Cooling the block by ice →
4. Disposable Embedding Mold Cutting the tissues and making ribbons of tissue
○ Ito sila usually ginalabay. → Floating the tissue in Water bath → Picking up
○ Peel Away, Plastic Ice Tray, and Paper the tissue on slide and drying
Boats

*Yung paper boat na box or rectangle tignan, hindi yung literal

na bangka
OTHER EMBEDDING METHODS
1. Double-Embedding Method
○ Tissues are first infiltrated with Celloidin and
subsequently embedded in a paraffin mass
TRIMMING
○ For large blocks of dense tissues
● Removal of excess wax using knife or cutter after
○ Since it’s not used nowadays, it’s considered
the wax block is removed from the tissue cassette or
obsolete.
paper boat
2. Plastic Resin Embedding
● There is a formation of a truncated pyramid and
○ For high-resolution light microscopy of
exposure of the tissue surface for ease of sectioning
thinner than usual sections, renal biopsies,
● Allow tissue blocks to fit into the block holder of
and BM biopsies (Bone Marrow).
microtome
○ Examples:
● At least 2 mm should surround the tissue block
■ Epoxy
○ Dapat may naiwan pang wax na 2 mm para
■ Polyester
at least it could provide a better trimming
■ Acrylic Plastic
function sa atin when we do trimming.
EPOXY
● Coarse Trimming
● For electron microscopy

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○ When we say coarse trimming, basically ito predetermined distance towards the knife for
yung pag cut ng tissue block, yung ating cutting sections at uniform thickness.
wax, parang gina pa expose mo pa lang ● Essential Parts:
siya, yan yung coarse trimming. 1. Block Holder
● Fine Trimming 2. Knife Carrier and Knife
○ There are two ways to do fine trimming, it 3. Pawl, Ratchet Feed Wheel and
can be done by either setting the thickness Adjustment Screws
adjuster at 15 mm or by advancing the block
using the coarse feed mechanism.
● So basically, mauna ang coarse trimming, at kapag
malapit na ma expose yung specimen natin, dun na
tayo gagamit ng fine trimming.

TYPES OF MICROTOME
1. Rocking or Cambridge
2. Rotary
3. Sliding
4. Freezing
5. Cryostat or Cold Microtome
6. Ultrathin
MICROTOMY
ROCKING MICROTOME (CAMBRIDGE)
● When the tissue is exposed, dito na papasok ang
● Simplest form of microtome
sectioning.
● Invented by: Paldwell Trefall (1881)
● AKA Sectioning
● 10-12 μm thickness (tissues are cut in slightly curved
● A process whereby tissues are cut into uniformly
planes thus not recommended for serial sections)
thin slices or "sections" with the aid of a machine
● Heavy base, 2 arms
(Microtome), to facilitate the studies under the
○ Lower arm - resting on pivots and
microscope.
supporting medium, attached to the
● Sections usually form ribbons due to slight heat
micrometer screw.
generated between the block and the knife edge
○ Upper arm - carrying the block holder on
during the process of cutting.
one end by means of a screw, is connected
○ Pag Sinabi nating ribbon, ito yung parang
to a lever by a piece of nylon thread.
tuloy-tuloy na pagdikit-dikit ng mga sections.
Kapag multiple sections na joined
together is what we call a ribbon.
● Incomplete sections are discarded; it should be
discarded kapag incomplete kasi di maganda na
kulang ang section, kasi kapag half lang ang section,
ibig-sabihin hindi niya nakuha ang buong mukha ng
specimen, so uulitin sya.
● Careful din when we do trimming kasi hindi pwede
mag-over trim kasi baka wala na matitira sa specimen
or baka yung part na natanggal natin is nan dun yung
mga cancer cells na supposed to be babasahin ng ROTARY MICROTOME (MINOT)
pathologist. ● Most common
● Complete ribbons are picked up with camel hair ● Used for routine and research laboratories
brush, forceps or fingers. ● Media: Paraffin
● Principle: ● Excellent for serial sections
○ Spring-balanced or pawl is brought into ○ Because the thickness is much thinner than
contact with, and turns a ratchet feed wheel the rocking microtome, so makakagawa ka
connected to a micrometer screw, which is in talaga na tuloy-tuloy na ribbons.
turn rotated, moving the tissue block at a ● Invented by: Minot (1885-86)
● Electrically driven is now available

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● Knife and block holder are brought together by

upward and vertical motions, cutting sections in a
perfectly flat plane.
● Relatively dangerous; because it utilizes disposable
blades, which are sharper compared to the traditional
microtome knives.

CRYOSTAT OR COLD MICROTOME

● More common than freezing microtome
● Used for rapid preparation of urgent tissue biopsies
for intraoperative diagnosis (STAT); STATIM or rapid
SLIDING MICROTOME turnaround time
● Developed by: Adams (1789) ● It consists of microtome - Rotary Microtome - kept
● Types of Sliding Microtome inside a cold chamber
1. Base-Sledge Microtome - electrically driven ● Chamber: -5 to -30°C; Average: -20°C
and ideal for resin-embedded decalcified ● Thermostat: capable of freezing fresh tissue within
bone. 2-3 minutes
2. Standard Sliding Microtome - knife is ● Cutting Section: 4 μm
moving (Most dangerous type of microtome) ● All the controls to the microtome are operated from
● Both Microtome, the knife can be set obliquely for the outside the refrigerated cabinet
celloidin or straight large paraffin ● Testing: Fluorescent antibody staining technique or
● Recommended for cutting "extremely" hard and histochemical enzyme studies
rough tissue blocks.
● Considered the most dangerous type of microtome
because compared to other microtomes wherein the
block is the one moving, the blade is the one moving
in a sliding microtome.

ULTRATHIN MICROTOME

FREEZING MICROTOME
● Invented by: Quickett (1848)
● Stage for block holder: Hollow and Perforated,
attached to a flexible lead pipe containing carbon
● Cutting section: 0.5µm
dioxide.
○ But there are some ultrathin microtome that
● Carbon dioxide - freezing agent
can cut at 0.1µm
● This type of microtome is firmly attached on the edge
● Media: Plastic (Resins, and etc.)
of the bench.
○ Hindi mo talaga magagamit kung walang
● For Electron Microscopy, tissues fixed with osmic
acid
edge yung table or bench niyo.
● Used to cut undehydrated tissues in a frozen state ● Uses special diamond knife
● ADV: Very sharp and no easy dulling

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INFILTRATION, EMBEDDING & SECTIONING

ii. Cryostat procedure (cold

So, tandan natin diba during sectioning, before ka talaga mag microtome)
sectioning you’re going to perform trimming. In Ultrathin
MICROTOME KNIVES
microscope guys, ang ginagamit na knives for trimming ay
Glass knives, while for sectioning ay diamond knives.
PLANE CONCAVE BICONC PLANE-WE
CARE OF THE MICROTOME
AVE DGE

LENGTH 25mm 120mm 100mm

CHARACTER One side is flat, Both Both sides

ISTICS other side is sides straight
concave concave

EMBEDDING Less More paraffin Paraffin

● Brush away accumulated paraffin and small MEDIUM concave concave
pieces of tissues with soft brush after sectioning
○ Why so? Because kapag hindi malinis yan, celloidin paraffin
una magdidikit-dikit ang ating tissue ribbons,
pangalawa, yung mga tissue might cross MICROTOME sliding Base-sl Rotary Base-sledge
contamination with the specimen na cinu-cut edge,
mo. rotary or
○ Excess paraffin and tissues may later on microto
interfere with the cutting of tissue blocks. me
○ Xylene — may also be used for cleaning
some parts of the microtome.
● Oil Movable Parts to prevent rusting.
● Cover microtome to prevent accumulation of dust.

TYPES OF TISSUE SECTIONS

1. PARAFFIN (4-6 µm)
a. Successive sections will usually stick
edge-to-edge KNIFE ANGLES
b. Sections are removed in ribbons of ten to
allow easy location of serial sections.
2. CELLOIDIN (10-15 µm)
a. The blocks are trimmed in the same manner
as in paraffin blocks
b. To avoid dehydration and shrinkage,
sections are usually cut by the wet method,
both the sections and the block being kept
moist with 70% alcohol during cutting.
c. Celloidin sections do not come off in ribbons
and have to be collected into 70% alcohol
immediately.
3. FROZEN SECTIONS 1. Bevel Angle: 27 ° to 32°
a. Methods of preparing frozen section a. Angle between the cutting edges
i. Cold knife procedure 2. Cutting Angle (14°) — angle between the upper
1. Basically, fresh yung facet of the knife and the surface of the tissue block.
specimen tapos yung knife 3. Clearing Angle (Clearance) — angle between the
lang talaga ang ipaexpose lower facet of knife and the surface of the tissue
mo sa cold temperature block: only angle that can be adjusted on microtome
and diretso mo siya cut sa using the knife tilt.
specimen.

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INFILTRATION, EMBEDDING & SECTIONING

a. Should be inclined at 5-10° from the cutting

plane so that the cutting facets will not
compress the block during the process of
cutting.
b. If too small, the body of the blade will scrape
the block, and skipped sections or poor
ribboning will result.
c. If too wide, the tip of the blade will scrape the
block and chatter will result.
OTHER KNIVES AND BLADES

1. Disposable Blades ● Widely used now

because cheaper;
honing and
stropping are no
longer common
practices. SHARPENING
● Coated with ● Badly nicked knives with blunted ends have to
polytetrafluoroethyle undergo sharpening in order to ensure optimum
ne (for ease of sectioning of tissue blocks.
ribboning) ● Sharpening of the knives involves 2 stages, namely:
● E.g. Magnetic
knives in cryostat HONING STROPPING

2. Glass knives/Ralph ● For Ultrathin

Removal of gross nicks Removal of burr/irregularities
knives microtomes

3. Diamond Knives ● For resin blocks on To acquire an even edge Final polishing of the knife
ultrathin microtomes; edge
brittle and expensive
HEEL to TOE TOE to HEEL
4. Safety razor blades ● For partially calcified
materials, paraffin HONING
and frozen sections.
● Easily replaced
HONES
when dull and
Natural sharpening KNIFE
produces good
stone or hard grinding LUBRICANTS SHARPENERS
tissue sections same
surface
as with microtome
knives.
Belgium yellow ● Soapy water Flat glass plate
● Unsatisfactory for — most common; ● Mineral Oil with finely
sections less than best result ● Clove Oil powdered
10 micro ● Xylene aluminum oxide
Arkansas ● Liquid – maybe used for
— has more polishing paraffin grinding and
effect removing nicks.

Fine Carborundum Diamantine — for

— coarser; for badly final polishing
nicked knives

Plate Glass 8x3x1in)

— excellent

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2. Knife is laid obliquely on the strop and with the cutting

edge behind
3. Edge last is pushed backward and drawn forward
ADHESIVES
● Used for adhesion of the tissue to be examined to the
slide
● For: Brain sections, decalcified tissue, and when
using strong alkali at time of staining.
● Mayer’s Egg Albumin — Most common: never used
with pap staining method as it will retain OG stain:
produces background staining/
○ Egg white — main adhesive
○ Glycerol – increase viscosity and prevents
drying
○ Thymol crystals — prevents mold formation
● Dried Albumin
PROCEDURE: ● Starch paste
1. Clean hone with xylene to remove scattered particles ● Plasma — outdated blood stored in blood banks,
of stones and metal dispensed into sterile tubes of 0.5 ml each.
2. Cover with lubricant ● 1% Gelatin - Better adhesion than albumin; heated
3. Knife is fitted to its corresponding back, placed on one prior to using: added to floatation bath; stains with
end of the hone with cutting edge first many dyes
4. Heel to Toe movement, Edge first: With cutting knife ● 1% Methyl Cellulose - does not retain the stain
edge first, the "heel" (handle end) is drawn obliquely ● Poly-L Lysine - no background staining: Most
or diagonally towards the operator on the stone until commonly used IHC adhesive
the "toe" (head portion) is reached. ● Sodium silicate - Strong adhesive: slight dye
5. Continue until all the teeth in the knife edge have retention, not affected by mild alkaline solutions.
been eradicated o DADV: Blackening in silver impregnation
6. Wash with water after using to remove the metal staining, reticulin methods, red staining
collected during the process in methyl green pyronin technique
7. After honing, wipe off the oil or soap from the knife o Resins - diluted 1:10 with acetone; Has
with xylene, then strop it thoroughly.... the greatest adhesion; One kind is
STROPPING araldite (made of epoxy resins)
FLOTATION / FLOATING OUT

Paddle strop made of horse leather

● Attached to a solid back, in order to prevent sagging o Sections are floated out on a water bath set at 45-50c
● Usually dry thus require oiling ● Flotation Bath
● Vegetable oil (e.g., castor oil) applied on the back of ○ 5 to 10°C MP (arrow down) of Wax
the horse leather ○ Inside is specifically colored enamel blạck
■ TSEs flatten after 30 sec; removes
● Not mineral oil because it tends to blister and destroy
the wrinkling
the leather
● Regulated temp. to flatten the sections and prepare
Procedure: Toe to Heel movement, Edge Last
them for mounting into the slides/slider
The procedure is the reverse of honing
● Selected sections for staining should to fish-out in a
1. The knife is fitted with its appropriate knife back
vertical position

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DRYING THE SLIDES

● Mounted sections are placed in a paraffin oven to dry.

● Hot plates are not recommended because they can
cause:
○ Overheating
○ Dust falling - onto the section during drying
period.
● Metal racks with 25-slide divisions are used to store
the mounted sections during the drying process which
usually takes 5 minutes in the heated oven. Once dry,
the whole rack of slides can be taken for manual
staining.

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