HSTCYT - EMBEDDING, MICROTOMY, STAINING, MOUNTING

Embedding/Blocking

This will come after the process of infiltration

Embedding; Tissue blocks

The process where you will be going to surround/solidify/secure your tissue into a structure known as __?__

Tissue Block

This will aid you in the creation of tissue ribbon during the microtomy process.

TRUE

TRUE OR FALSE

In microtomy, in frozen tissue sectioning or fresh tissue sectioning, you cannot create a ribbon/perform microtomy but rather a singular section out of tissue specimen is created because the embedding process is omitted.

block holder

This is necessary so that your tissue block would not move around.

Infiltrating and embedding

these two processes will reinforce your tissues to such a way that your morphology of the tissue are kept preserved especially in the microtomy process.

strength and hardness

Ideally the embedding medium should match the tissue type in ___ and ___

Orienting

A process where tissues are placed in a certain way inside the cast to demonstrate the required structure on the tissue section.

cassettes

Tissues are placed in ___ in a way that they must be placed in the tissue block.

cross section

Seeing the different layers of different organs thus this orientation is necessary whenever you are going to encast your tissues.

EMBEDDING MOULDS

Cast with a pre-set shape of rectangle or square

Allows the medium to cool and solidify according to the shape of the recess of the mould and according to the material of embedding mould.

FALSE

faster or slower

TRUE OR FALSE

Embedding moulds will either allow slower cooling process

metals, brass, steel, or aluminum

These embedding moulds are materials that is good conductors or dissipater of heat

Trimming

Depending on the type of mould, blocks may require ___ so that it will fit inside the tissue block holder of the microtome.

Paper boats

Disposable embedding cast that are utilized for embedding celloidin blocks but are equally useful for paraffin wax blocks.

Peel-away

Disposable embedding cast that is made from paper or cheap type of disposable plastic which can be disposed after it had been used or cannot maintain its shape

Plastic Ice Trays

This used in the laboratories when creating several tissue blocks

Advantage: can encast multiple tissue samples.

Leuckhart's Embedding Mould (L mould)

Blocks produced are even, with parallel sides, and with a shaped initial setting of the wax.

2-piece brass, metal, aluminum, or wood L shape mould

The mold is adjustable to give a wide variety of sizes to fit the size of the tissue block for casting

It is recommended for routine use, although, too slow and cumbersome for use in a busy laboratory.

Compound Embedding Unit

Has an advantage that is the same with plastic ice trays which can be used where there are multiple tissues to be embedded.

Disadvantage: Most expensive

Tek system

Plastic Embedding Rings and Base Moulds that includes ease of use, less paraffin wax needed, faster embedding, firmly attached tissue and holder, and permanent identification.

It produces easier orientation when resectioning of tissue is required, and blocks can be filed immediately after sectioning

PLASTIC EMBEDDING RINGS

Have a preset shape that could fit some or not most of

the tissue blocks

Advantage: No need to trim the tissue blocks

TISSUE CASSETTES

A device that is helpful during the orienting process

Will hold the tissue processing all the way to infiltrating

process

Not used as moulds

blocking

Whatever the infiltrating medium used, the same material is used for ____.

dense embedding

_____ medium is necessary for blocking tissues to be cut with a heavy microtome knife.

2-mm

In embedding, There must be at least a ___ gap between the outline of the block and the tissue.

5C - 10C

Embedding media are often at its molten state, and are maintained at a temperature ____ higher than its melting point.

cooled; refrigeration

After placing the tissue in the embedding medium, it is ___ via ____ of is soaked in a cool water for it to solidify.

small; to cut

Micros and temnein means

MICROTOMY

Process of UNIFORMLY cutting embedded tissue specimens into thin slices (sections), forming RIBBONS

Allows study of tissues at a microscopic level

Microtome

Device that cuts tissue blocks into thin sections

Block Holder (Chuck)

Part of Microtome that:

Will hold the block in place

May vary in terms of design depending on

embedding moulds used

Knife holder/carrier

Part of Microtome that:

hold the knife in place whenever we cut the tissue specimen

Pawl, Ratchet Feed Wheel, and Adjustment Screws (Mechanical/Moving Parts)

To line up the tissue block in proper position

with the knife, adjusting the proper thickness

of the tissue for successive sections.

Pawl

Part of Microtome that:

prevents the ratchet feed wheel

rotating to the opposite direction

Adjustment screws

allows adjustment of certain aspects of the microtome such as when adjusting the thickness of the section we are creating

ROTARY

Minot (1885-1886)

2-4 μm; 3-5 μm

Type of Microtome:

Most used in clinical and research laboratories

Actual cutting is bases on the rotary action, cranking the flywheel

Can cut specimen with ___ (Bruce-Gregorios ___ (will be used as the main reference)

Advisable for paraffin and celloidin-embedded tissues

SLIDING

Adams (1789)

> 3μm

Type of Microtome:

Ideal for cutting sections with thickness ___ than ___

Most dangerous type; exposed moving blade

Standard Sliding

Type of Sliding Microtome:

The block remains stationary while the knife glides over

Good for celloidin-embedded tissues

Most dangerous as the knife is moved instead of the block

TRUE

TRUE OR FALSE

One importance of embedding is to provide an additional support during the microtomy process apart from creating ribbons.

FALSE

cut uniformly

TRUE OR FALSE

Encasing an infiltrated tissue sample in a medium while in a cast (mould) that will aid in the microtomy process. Embedding also ensures that tissues are cut varied and will minimize the tendency of destroying the entire tissue sample.

Base-Sledge

Type of Sliding Microtome:

Consist of 2 pillars which holds the knife; specimen passes below the blade

Ideal for sectioning tough or large blocks with > 10 μm thickness

Originally designed for sectioning very large

blocks

Heavier; more stable which is not influenced by any environmental vibrations

ROCKING (CAMBRIDGE)

Paldwell Trefall (1881)

Type of Microtome:

Simplest

Lever-action

Sections are cut in a slightly curved plane, with 10-

12 μm thickness

Not recommended for creating serial sections

because of the slightly curved plane result

FREEZING (QUECKETT)

Queckett (1848)

Type of Microtome:

Block holder is hollow and perforated, and is

attached to a reinforced flexible lead pipe

It is used for frozen section specimen

Ideal for fat and neurological (Myelin sheath in

spinal cord) specimen cutting, also for tissues easily destroyed by heat which allows you to perform IHC and immunofluorescence

Carbon dioxide gas (Most commonly used)

Liquid nitrogen

Isopentane (Most preferred to be injected)

Freezing Microtome is maintains their cold temperature due to these different coolant agents:

CRYOSTAT (COLD MICROTOME)

rotary rocking

5C - -30C

Type of Microtome:

Consists of a refrigerated chamber that freezes tissues to a correct degree of hardness

Contains a ____ microtome

Chamber is maintained at a temperature of ___ - ____

(Average of -20C)

Ideal for preparing thin sections of fresh frozen tissue

sections

Produces 1 section at a time since no tissue is

embedded

ULTRATHIN MICROTOME

60-100

0.5 - 1

Type of Microtome:

Cut tissue for electron microscopy

Uses a glass or gem-grade diamond knife to cut

sections with ____ μm thickness

Semi-thin sections with ___ μm thick can alsi be produced when a glass knife or an industrial-grade diamond knife is equipped

stainless steel

(some are made from tungsten carbide)

Knives are made from?

PROFILE A (PLANOCONCAVE/PLANE-CONCAVE)

Knife profiles:

25 mm length

Used on both sliding and base sledge, rotary, or

rocking microtomes

PROFILE B (BICONCAVE)

Knife profiles:

120 mm length

Recommended for cutting paraffin sections of rotary microtomes

Because of its 2-concave side, it is the least stable type of microtome knife because of the vibration while cutting

PROFILE C (PLANE-WEDGE/WEDGE)

Knife profiles:

100 mm length

Frozen sections

Extremely hard and tough specimens in paraffin

(using base sledge)

PROFILE D (TOOL EDGE/CHISEL)

Knife profiles:

Plain sides with a steep edge

For cutting very dense tissues (undecalcified bone)

Hard to hone

Polytetrafluoroethylene

Disposable knives are coated with ___ to create tissue ribbon without sticking to the knife

Low-profile

Profile of disposable knife used for cutting soft tissue; large but soft tissues

High-profile

Profile of disposable knife used cutting firm and relatively hard tissues but not for undecalcified bone specimens

GLASS AND DIAMOND KNIVES

Incorporated in ultrathin microtomes

Produces the thinnest type of sections

Glass

These knives are cheap; easily replaced

For very hard tissues

Diamond

These knives are expensive; relatively hard to sharpen

For epoxy resin-embedded tissues

WATER BATH (FLOATATION CHAMBER)

Thermostat

distilled water

surface tension

For floating tissue sections after cutting

Set at a warm temperature, often controlled by a

____ to flatten out the cut section

Preferably, ___ must be used

Adding alcohol or a small drop of detergent/soap

helps in reducing ____

Forceps

used to transfer the tissue ribbon from the microtome to the water bath

Brushes

used to clean unnecessary debris fallen in the microtome

Needles such as inoculating and applicator sticks

can be used for transferring tissue ribbons

DRYING OVEN/HOT PLATE

37 C

Designed with a fan inside the oven to circulate the hot heat inside

For drying slides and for deparaffinization process

For delicate specimen, it is better to use lower temperatures (___for 24 hours)

ADHESIVES

Prevents tissues from detaching from the slide

0.1% Poly-L-lysine

ADHESIVES

diluted further to a 1;10

solution for IHC

2% 3-aminopropyltriethoxysilane (APES)

ADHESIVES

for bloody or proteinaceous specimen; cytological

studies

thymol crystal

phenol

ADHESIVES

protein adhesives must be mixed with ___ to act as anti-microbial agent. For carbohydrates-based adhesives, mix it with __

SECTIONING

The process in where you will be producing tissue sections from tissue blocks

Paraffin Sections

Cutting of paraffin embedded tissue blocks with rocking and/or rotary microtome

Celloidin Sections

Cutting of celloidin embedded tissue blocks

with sliding microtome

Frozen Sections

Creation of frozen tissue section and sectioned using either cryostat or freezing microtome

5-7

2-4

Tissue block that are relatively dense - adjust it to around ___degrees

Tissue block that are relatively soft - adjust it to ___ degrees

RAKE ANGLE

Angle between the bevel of the knife and the imaginary perpendicular line from the surface of the blocks

CUTTING ANGLE/BEVEL ANGLE

27-32

Angle formed from the cutting edges of the knife

___ degrees

CLEARANCE ANGLE

5-10

15

Angle formed from the surface of the block to the cutting facet

____ degrees prevents uneven sections

____ degrees allows better penetration to the tissue,

minimizing sections

chuck

Prior to trimming, the ____ must be retracted

COARSE FACING/COARSE TRIMMING

This trimming may be

done to expose the tissue sample

moth hole

Aggressive trimming can cause _____ artifacts. A small piece of the tissue will be removed together with the wax resulting now to a hole when viewed under microscope

FINE TRIMMING

This trimming makes the face of the tissue block smoother, for creating better tissue sections

Chilled or cold

_____ paraffin blocks can provide better sectioning

Cooling

5-10

This should be done ___ min (Bancroft); 10- 20 min (Dey) which would help of the expansion of the tissue specimen because of the formation of moisture in the surface of the tissue block.

teasing

Folds and creases may be removed by gently

____ the sections

TRUE

TRUE OR FALSE

Sections must not be left on the water bath for 30

seconds

tissue paper

Debris present in the water bath must be removed through dragging ____.

wet stone and leather strop

Materials needed for knife sharpening

Nicked knives

These are possible because of cutting bones which was not undergone complete decalcification

HONING

Removal of gross nicks on the knife edge

(Coarse) - repairing the knife itself

Grinds the edge to create an even edge (Honing

Proper)

STROPPING

Removes burrs that form during the sharpening process

Polishes the knife with leather strop

Burrs

These are product of honing of knife

FINE CARBORUNDUM

Honing stone:

Coarsest of all the whetstones (low grit - granule content)

For repairing badly-nicked knives, followed by honing with a Belgian Yellow stone or Arkansas stone

Can also be used for coarse honing

Commonly used in kitchen knives

BELGIAN YELLOW/BELGIUM COTICUL

Honing stone:

Less coarse vs. carborundum

Provides a better polish after honing

Best results for honing

For blunt or nicked edges

ARKANSAS

Honing stone:

Provides a better finish out of all the stones

Better polish vs. Belgian yellow

Because of its smooth surface, you can use it for fine

honing procedure

xylene

HONING PROCESS

Surface of the stone is cleaned with a soft cloth moistened with ____

HEEL to TOE fashion

20-30

HONING PROCESS must be done in what fashion?

Must be done ___ strokes on each side

vegetable

STROPPING PROCESS The strop is oiled using ___ oil applied on its back

toe to heel pattern

40-120 strokes

STROPPING PROCESS must be done in what fashion?

Must be done ___ strokes on each side

Done typically for plain wedge knife

STAINING

Process of introducing an unstained specimen to a dye

FALSE

Affinity

TRUE OR FALSE

Stains add contrast to the different structures of tissues, depending on their avidity to the stain and the intensity of the stain.

FALSE

not the real color of a tissue

TRUE OR FALSE

The colors of stains

are the real color of a particular tissue.

TRUE

TRUE OR FALSE

Cells are transparent and colorless

FALSE

lacks natural pigments

TRUE OR FALSE

Tissue specimen lacks artificial pigments that is why it is colorless