TISSUE FIXATION

 Fixation is a complex series of chemical events that differ for the

different groups of substance found in tissues.
 The aim of fixation:
1- To prevent autolysis and bacterial attack.
2- To fix the tissues so they will not change their volume and shape
during processing.
3- To prepare tissue and leave it in a condition which allow clear
staining of sections.
4- To leave tissue as close as their living state as possible, and no small
molecules should be lost.
 Fixation is coming by reaction between the fixative and protein which
form a gel, so keeping every thing as their in vivo relation to each other.
 Factors affect fixation:
- PH.

- Temperature.

- Penetration of fixative.

- Volume of tissue.
According to previous factors we can determine the concentration of
fixative and fixation time.

Types of fixative:
Acetic acid, Formaldehyde, Ethanol, Glutaraldehyde, Methanol and Picric
acid.
 TISSUE PROCESSING
the aim of tissue processing is to embed the tissue in a solid medium
firm enough to support the tissue and give it sufficient rigidity to
enable thin sections to be cut , and yet soft enough not to damage the
knife or tissue.

Stages of processing:
1- Dehydration.
2- Clearing.
3- Embedding.
Dehydration
to remove fixative and water from the tissue and replace them
with dehydrating fluid.
There are a variety of compounds many of which are alcohols.
several are hydrophilic so attract water from tissue.
 To minimize tissue distortion from diffusion currents,
delicate specimens are dehydrated in a graded ethanol
series from water through 10%-20%-50%-95%-100%
ethanol.
 In the paraffin wax method, following any necessary post
fixation treatment, dehydration from aqueous fixatives is
usually initiated in 60%-70% ethanol, progressing through
90%-95% ethanol, then two or three changes of absolute
ethanol before proceeding to the clearing stage.
Types of dehydrating agents:
Ethanol, Methanol, Acetone.

 Duration of dehydration should be kept to the minimum consistent

with the tissues being processed. Tissue blocks 1 mm thick should
receive up to 30 minutes in each alcohol, blocks 5 mm thick require up
to 90 minutes or longer in each change. Tissues may be held and stored
indefinitely in 70% ethanol without harm
Clearing
 replacing the dehydrating fluid with a fluid that is totally miscible with
both the dehydrating fluid and the embedding medium.
 Choice of a clearing agent depends upon the
following:
- The type of tissues to be processed, and the type of processing to
be undertaken.
- The processor system to be used.
- Intended processing conditions such as temperature, vacuum and
pressure.
- Safety factors.
- Cost and convenience.
- Speedy removal of dehydrating agent .
- Ease of removal by molten paraffin wax .
- Minimal tissue damage .
 Some clearing agents:
- xylene.
- Toluene.
- Chloroform.
- Benzene.
- Petrol.
Embedding
 is the process by which tissues are surrounded by a medium such as
agar, gelatin, or wax which when solidified will provide sufficient
external support during sectioning.
 Paraffin wax
properties :
 Paraffin wax is a polycrystalline mixture of solid hydrocarbons
produced during the refining of coal and mineral oils. It is about two
thirds the density and slightly more elastic than dried protein. Paraffin
wax is traditionally marketed by its melting points which range from
39°C to 68°C.
 The properties of paraffin wax are improved for histological purposes
by the inclusion of substances added alone or in combination to the
wax:
- improve ribboning.
- increase hardness.
- decrease melting point
- improve adhesion between specimen and wax
Clearing Agents :

1- Xylene= Xylol

Commonly used in clearing, its boiling temperature is about

140C, cheap.
Very effective, make tissue transparent , and can be removed
easily during infiltration .
While some of its deformities are causing shrinkage if tissues
and hardness if they left for along time.
It is not suitable for clearing both of brain and lymphatic
tissues because it make them fragile.
It appears as milk color if the specimen contain some of water.
2- Cedar- wood oil:

The light types of it used for clearing purpose, its

boiling temperature range between (168-237) C.
It dose not cause any hardness for tissues even they left
for along time and easily penetrating tissues.

While, it is consider expensive and it needs very long

time to get it out from tissue in wax oven. ( The wax
should be changed for three times in each use).
3- Benzine:

Its boiling temperature 80 C. It’s easily penetrate tissue,

not make them soft or fragile, never cause any atrophy
to tissues.
Evaporate quickly in wax oven, cheap, but it is highly
ignition. Its vapor cause poisoning even in low
concentration.
4- Chloroform:

Its boiling temperature 61 C. It has a little effect on

tissue for both shrinkage and hardness in comparison
with xylene.
It consider excellent clearing for embryo, nervous
system and lymphoid organs.
Quickly evaporated at wax oven.
It doesn't ignite, and very expensive.
5- Toluene= Toluol:
Its boiling temperature 110 C. Better than Xylene.
Because of its little concretion for tissues.
While its clearing for tissues not as xylene and benzene.
There another clearents such as Clove oil, Turpinol , and
Methyl Salicylate.
All clearents added on tissue at ratio 1:10 from tissue
size. Incomplete clearing cause disturbance in work and
make tissue dark, also cause milk color for them.
The change of clearent depend on size and type of
tissue.
Infiltration and Embedding Agents:

The purpose of all steps previously is to prpare tissue

for infiltration then embedding in paraffin wax
- Paraffin wax
- Paraplast
- Carbowax
- Synthetic Resins
- Celloidin
- Gelatin
- Agar
TISSUE SECTIONING
tissue preparation
A small piece of the targeting organs (brain,
liver, kidney, lung and skin) .
1- Fixation: small pieces of the targeting organs
of the infected mice
or rats were kept in 10% formalin for 24 hours.
2- Washing: tissue samples were washed in
water for 20 min to eliminate the access of
fixative.
3- Dehydration: the samples were passed at a
graded series concentrations of ethanol (70, 80,
and 90%), 2 hours each concentration, and then
left in 100% ethanol, changed twice, for an hour
each time.
4- Clearing: the tissue clearing was done by( •
chloroform, benzin ,xylen ) for 12 hours .
5-Wax infiltration: tissue samples were placed in
melting paraffin at 60C for 8 hours in the oven.
6-Emmbedding: the wax from the last step was
discarded and the tissue samples were
transferred quickly to base mold at the right
direction, melting paraffin was added and left to
harden at room temperature.
7-Sectioning: wax blocks were trimmed and
fixed on rotary microtome on the right direction
to obtain 5-7 microns thick slice. The separate
tapes of sections were transported to 45C water
path then loaded on glass slides covered with a
smear of albumin Mayer. The slides were kept
on hot plate at 45C in order to flatten the
sections, then they left at room temperature for
24 hours before staining.
8-Mounting and staining: the tissue
sections were stained using dye
hematoxylin-eosin and mounting by
DPX and cover slip were added.