Dehydration

Removal of water from tissues by using descending

.grades of Alcohol

.Purpose: 1- prevent tissue's rotting

.Prepare tissue for the next steps -2

Other dehydrants can be used, but have major

.disadvantages
.Acetone is very fast, but a fire hazard
Dioxane can be used without clearing, but has
.toxic fumes
 Dehydration
:Procedure

Time for each concentration of alcohol= 30

.minutes to 1 hour
 Clearing
Removal of alcohol with a substance that will be
.miscible with the embedding medium (paraffin)
.The commonest clearing agent is xylene
Purpose: preparing the tissue for infiltration
? step. Why
Toluene works well, and is more tolerant of
small amounts of water left in the tissues, but is
.3 times more expensive than xylene
Chloroform can be used, but it is a health
.hazard, and slow
 Clearing
:Procedure

Note: Excessive exposure to clearing reagents

.may cause excessive hardness or shrinkage
 Impregnation( Infiltration)
Immersion the samples in hot paraffin inside the
.oven

The paraffin will penetrate in-between the *

.cells of the tissues

Purpose: This process of paraffin infiltration is a

necessary step to harden the tissues before their
.embedding
 Impregnation
:Procedure
Bring 4 beakers then put proper amount of -1
liquid paraffin wax in each breaker then label
.the beakers (wax1, wax2, wax3, and wax4)
.put all beakers inside the oven -2
put the samples in wax1, wax2, wax3, wax4 -3
. respectively for 1/2 h

Note: Infiltration must be carried out at only two

.degrees above the melting point of paraffin
 Embedding
The processed tissue is orientated within a
metal mould which is then filled with molten
.paraffin wax

Purpose: support the tissue Sample from

.outside
 Sectioning
sectioning the tissue blocks using a rotary
.microtome
The paraffin embedded tissue block is placed in-
the microtome and the wax trimmed away until
.the tissue is exposed

Purpose: harden tissues, in order to be able to

.cut suitably thin sections for microscopy
 Types of microtomes
A microtome is an instrument used to make thin
slices of tissue (usually 4 μm but can be 2–10
.μm)

:Types
Rotary Microtome -1
Ultraviolet Microtome -2
Cryostat -3
:Microtome essential components
Base/body -1

Knife and knife holder -2

Block Holder -3

thickness adjustment -4
 Mounting
.Is putting the cut tissue over a water bath
Purpose : To eliminate wrinkles and distortion
.in the tissue, and picked up on a slide

The temperature of the water bath depends on

the type of wax and is typically 5–9 °C below the
.melting point for the wax
Mounting and Drying
 Staining
Hematoxylin and Eosin (H&E) staining technique
is used to stain the nuclei blue to purple
(hematoxylin), and counterstain the cytoplasm
.pink to red (eosin)
Staining and Covering