13/9/2011

Histological Techniques:
TISSUE PROCESSING FOR HISTOLOGY
.

Siti Fathiah Masre (MSc.)

Biomedical Science

Histology
Histo = tissue ; Logy = study
study of tissue

Objectives:
Examination of minute structures
Examination for enzyme study
Examination of cell (cytology)
Examination of ultrastructure
Examination of structural changes

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PREPARATION FOR PROCESSING

TISSUE SAMPLES FOR HISTOLOGY

Suitable Fixative
Protocol
No. of Samples and labeling
Time Management

TISSUE PROCESSING PROTOCOLS

Steps In Tissue Processing:

1. Fixation
2. Embedding
- dehydration
- clearing
3. Sectioning
4. Mounting
5. Dewaxing

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FIXATION
To preserve tissue architecture
To prevent tissue from rotting or autodigestion

FIXATION
CHEMICAL FIXATION:
10% buffered formaline
10% formaline
bouin's solution
2% buffered gluteraldehyde
karnovsky

FACTORS AFFECTING FIXATION

Buffering to prevent tissue becoming acidic

Volume 10:1 (fixative : tissue)
Temperature
Concentration
Time interval - from tissue sampling to in the
fixative

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Fixation Steps:
1. Isolate the tissue into PBS as soon as possible.
2. Wash tissue with PBS to remove all blood.
3. Cut tissue to proper size. The size can be 5X5 mm
to 1X2 cm. The cutting surface of the tissue should be
flat and smooth.
4. Transfer tissue to fixative and swirl the container to
ensure all tissues are completely immersed in
fixative. The volume of fixative must be at least 10
times the tissue volume.

Fixation steps cont.

5. Fix tissue for overnight.
6. Check samples the next day to ensure proper
fixation.

EMBEDDING
To provide physical support to tissue can be
sectioned thinly.
Involved dehydration (alcohol) and clearing
(xylene) before hardening tissue by wax or resin.

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Embedding cont.
1. Dehydration with series of alcohol:
30% ethanol (2 hours)
50% ethanol (2 hours)
70% ethanol (overnight)
95% ethanol (3 hours x2)
100% ethanol (1 hour x2) .
2. Clearing in xylene (2 hours x2)
3. Infiltration in 60 wax paraffin (2 hours x3)
4. EMBEDDING in mold, oriented as specified

Embedding cont.
5. Pour hot wax paraffin in mold, tissue block
harden on cold surface

Embedding
machine

SECTIONING
1. Using machine called a microtome.
2. Ideal thickness of sections is 4 to 6 micron for light
microscopy.
3. The embedded wax block need to be placed on ice
and trimmed before sectioning.
4. Sharp blade is important to create a ribbon of
sectioned tissue.

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MOUNTING
1. Ribbon of sectioned tissue is placed onto
warm water to flatten them out.
2. Glass slide is placed towards the ribbon to
pick it up from water (fishing).

Dewaxing
To remove the wax from tissue in order for stain
to penetrate into the tissue. (mostly stains dont
mix with wax)
Dewaxing involved:
- sections dipped into xylene
- dipped into alcohol
- dipped into water
Proceed to staining

Summary
Fixation
Embedding (dehydration and
clearing)
Sectioning
Mounting
Dewaxing
Staining

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THANK YOU
TERIMA KASIH
KAMSAHAMNIDA