Technical Writing

Tissue processing
By
Lecturer
Noor Hashim Ismail
Cancer Research
Department
ICCMGR
Tissue processing
describes the steps required to take
animal or human tissue from fixation to
the state where it is completely
infiltrated with a suitable histological
wax and can be embedded ready for
section cutting on the microtome.
 types : manually or automated
Tissue processing steps
1-Fixation.
2-Sectioning
3-Dehydration.
4-Clearing (or de-alcohlisation).
5-Impregnation in wax ( Wax infiltration).
6-Embedding in paraffin block.
7-sectioning and slide preparation
8- staining
9- mounting
Fixation
Aiming to preserve a sample of biological
materials ( tissues or cells) as close to its
natural state as possible in the process of
preparing tissue for examination.
Factors affecting fixation:
PH , temperature, volume ,Time
interval
Sectioning
Each organ
or tissue has
a special
procedure in
sectioning.
Dehydration
• This process must be carried out so
that the embedding medium can
infiltrate properly.
• Alcohol in various dilutions is usually
used.
• water from the tissues should be
removed because water is not miscible
with wax
• prevent tissue shrinking.
Clearing
• As paraffin wax is insoluble in alcohol, it
must be replaced by a fluid that is
miscible with or is a solvent of paraffin
wax, to get rid of alcohol & make the
tissue more translucent.
• Xylol: cheap and rapid in action, though
tends to harden tissue on prolonged
application
Impregnation
(Wax infiltration)
• Tissues are impregnated in wax for two reasons:
1. To surround tissue with some plastic substances to support it
on all sides without injury.
2. To enable natural cavities of tissue to be filled with wax, thus
preserving their relationship to each other.
• Paraffin wax used for routine work
• Tissue may be impregnated with paraffin wax using:
▪ 56 C° oven or
▪ Vacuum embedding.
Paraffin for impregnation must be clean. (i.e. filtered).
Tissues which benefits most from vacuum embedding are:-
Lung, bone, fatty tissue, all tissues from CNS and lymph nodes.
•
Embedding
 Process by which tissues are surrounded by a
medium wax which when solidifies will provide
sufficient external support during sectioning.
 Impregnated tissue transferred from wax
bath to a mould filled with molten wax to get a
block of wax with the tissue specimen at the
center with the cutting surface facing the base
of the block
 This may be done using :
 L-shaped brass boxes.
 Embedding cassette
Sectioning
 Embedded tissues, to be
cut into thin sections of
3- 5 µ with a microtome.
 Cut sections are, floated
on a warm water bath
 Helps to spread the

specimen and remove
wrinkles.
 Floated sections are picked
up on an adhesive coated
glass slide.
 Glass slide kept on a slide
warmer at 580 temp for
20 min to ensure adhesion
 Egg albumin with additives
- commonly used adhesive
Staining
• Biochemical technique of adding a class-specific
dye to a substrate (DNA, proteins, lipids,
carbohydrates) to qualify or quantify the presence
of a specific compound
• Commonly used Staining procedures
– Gram staining
– Haematoxylin and eosin (H & E) staining
– Papanicolaou staining (PAP)
– PAS staining
– Masson's trichrome staining
H & E stain/ Haematoxylin &
Eosin stain
• Most popular staining method in histology.
• Most widely used stain in medical diagnosis.

• The staining method involves application of
the basic dye haematoxylin, which colors
basophilic structures with blue-purple hue,
and alcohol-based acidic eosin Y, which
colors eosinophilic structures bright pink.
Mounting
 The stained section on the slide must
be covered with a thin glass coverslip
to protect the tissue from being
scratched, to provide better optical
quality for viewing under the
microscope, and to preserve the
tissue section for years to come
 Mounting medium is used to adhere
the coverslip to the slide
 DPX & Canada balsam commonly used
Special techniques
• Immunohistochemistry
• Molecular pathology
• Microbiology
GIEMSA STAIN FOR H PYLORI
IHC FISH CISH

Thank you

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Tissue processing

4-Clearing (or de-alcohlisation).

5-Impregnation in wax ( Wax infiltration).

6-Embedding in paraffin block.

7-sectioning and slide preparation

 Helps to spread the

• Most widely used stain in medical diagnosis.

IHC FISH CISH

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