Dehydration

Removal of water from tissues by using

descending grades of Alcohol.

Purpose: 1- prevent tissue's rotting.

2- Prepare tissue for the next steps.

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:
1- Bring 4 beakers then put proper amount
of liquid paraffin wax in each breaker then
label the beakers (wax1, wax2, wax3, and
wax4).
2- put all beakers inside the oven.
3- put the samples in wax1, wax2, wax3,
wax4 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:
1- Rotary Microtome
2- Ultraviolet Microtome
3- Cryostat
Microtome essential components:
1- Base/body

2- Knife and knife holder

3- Block Holder

4- thickness adjustment
 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