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2. Tissue processing

The aim of tissue processing is to embed the tissue in a solid material 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.

The five main stages in the preparation of histological slides are:

• 1. Fixation

2. Dehydration

3. Clearing

4. Sectioning

5. Staining

 

2.1. Fixation

The aim of fixation is to preserve the tissue in a state that most reflect the living cell. Choice of fixative is generally dependent on the tissue of interest as different fixatives better preserve particular tissue elements.

Aim of fixation:

 i. To prevent autolysis and bacterial attack

 ii. To fix the tissues so they will not change their volume and shape during processing

 iii. To prepare tissue and leave it in a condition which allow clear staining of sections

 iv. To leave tissue as close as their living state as possible and no small molecules should be lost

 

2.1.1. Conventional chemical fixation

Chemical fixatives are used to preserve tissue from degradation, and to maintain the structure of the cell and of sub-cellular components, such as cell organelles (e.g., nucleus, endoplasmic reticulum, mitochondria). There are many fixatives which have been developed over the years using mixture containing heavy metals or picric acid, but the most commonly used fixatives for general plant histology are buffered aldehyde and formalin/acid/alcohol mixtures (FAA).

Aldehyde fixatives usually contain a combination of paraformaldehyde and glutaraldehyde: paraformaldehyde rapidly penetrates the tissue while glutaraldehyde gives superior cross linking. Higher concentrations of glutaraldehyde improve morphological preservation but excessive cross linking destroys delicate antigenic sites on proteins. FAA fixes nucleic acids very well but gives poorer morphological preservation and makes the tissue hard and, therefore, more difficult to section.