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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 auto-digestion

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