Tissue Preparation for Light Microscopy

What are the 5 stages of tissue preparation?

• Fixation

• Processing

• Embedding

• Sectioning

• Staining

Fixation

• What

• Aim

• Factors affecting fixation

• Types of fixation

What: A treatment with chemical agents to preserve it by slowing down natural breakdown, thus ensuring that the structure maintains close to original

Aim:

• Prevent autolysis and bacterial attack

• To fix tissue so volume and shape won’t change

• To leave tissue as close as their living state as possible

Factors affecting fixation:

• pH

• Temperature

• Penetration of fixative:size of tissue sample

• Volume of fixative:tissue

• Fixation time (24-48 hours)

Types:

• Bouin’s solution

• Acetic acid

• Formaldehyde 10%

• Ethanol

• Methanol

• Picric acid (bone)

Processing

• What

• Needs to be firm so

• But also needs to be soft enough because

• Stages

What: When tissue is placed in a supportive material (like paraffin wax) to hold it steady

Needs to be firm: So tissue can be sliced very thinly

But also needs to be soft: So it doesn’t damages the knife or tissue

Stages:

• Dehydration

• Clearing

• Impregnating

• Embedding

Processing: Dehydration

• What

• Uses

• Aim

• Delicate specimens are dehydrated in graded ethanol series:

What: Part of processing that removes fixatives and water from tissue and replaces them with dehydrating fluid

Uses: Hydrophilic compounds such as ethanol, methanol, acetone

Aim: Minimize tissue distortion from diffusion currents

Graded ethanol series: 10%-20%-50%-95%-100% ethanol

Processing: Clearing

• What

• Choices of clearing agent depends on

• Examples

What: When the dehydrating fluid is replaced with a fluid that is mixed well (miscible) with both dehydrating fluid and embedding medium

Choices of clearing agent depends on:

• Type of tissue

• Processor system used

• Safety factors

• Cost and convenience

• Speedy removal of dehydrating agent

• Ease of removal by molten paraffin wax

• Minimal tissue damage

Examples:

• Xylene

• Toluene

• Chloroform

• Benzene

Embedding

• What

• Most common wax

• Precautions while embedding

What: Process by which tissues are surrounded by a medium (agar, gelatin or wax) which when solidified provides sufficient support during sectioning

Most common wax: Paraffin wax

Precautions while embedding:

• Wax is clear of clearing agent

• No dust particles present

• Immediately after tissue embedding, wax must be rapidly cooled

  • To reduce wax crystal size

Embedding: Paraffin Wax

• What

• Characteristics compared to dried protein

• Melting point

• What it does to sample

What: Polycrystalline mixture of solid hydrocarbons

Characteristics: 2/3 density and more elastic than dried protein

Melting point: Ranges from 39C -68C

What it does to sample:

• Improves ribboning

• Increases hardness

• Decrease melting point

• Improve adhesion between specimen and wax

Sectioning

• What

• Equipment

• Best ribbon section size

• After sectioning:

What: To section solidified paraffin-embedded tissue

Equipment: Microtome and blade

Best ribbon section size: 4 to 5 µm

After sectioning:

• Spread tissue in flotation water bath at 42C

• Select best section before mounting

• Put into dryer hotplate at 37C overnight

Staining

• Routine staining consist of

• Special staining consist of

• Immunohistochemistry staining consist of

Routine staining: Hematoxylin and Eosin (H&E)

Special staining: PAS, Giemsa or fluorescent

Immunohistochemistry staining: Uses antibodies such as immunoperoxidase and immunofluorescent

Staining: H&E Routine Staining

• Principal

• Hematoxylin

  • Type of dye

  • Stains

  • Color

  • AKA

• Eosin

  • Type of dye

  • Stains

  • Color

  • AKA

Principal: Chemical attraction between tissues and dye

Hematoxylin:

• Type of dye: Basic

• Stains: Acidic structures like nucleus, chromatin, ribosomes

• Color: Purplish blue

• AKA: Basophilic

Eosin:

• Type of dye: Acidic

• Stains: Basic structures like cytoplasm

• Color: Red or pink

• AKA: Eosinophilic