The study of histology is to understand the microanatomy of cells, tissues, and organs and also to learn about their functions in structural terms.

FIXATION

Without question, the most important step to viewing biological tissue is fixation. The purpose of fixation preserves the structure of the tissue permanently in as life-like a state as possible.

Fixation should be carried out as soon as possible after the removal of the tissues or (if possible) in-vivo or soon after death (in the case of autopsy). A variety of fixatives is available for use depending on the type of tissue present and the features to be demonstrated. Check literature for optimum fixation methods for your specimen.

CHEMICAL FIXATION

A. Aldehydes: aldehydes fix samples by cross-linking proteins, especially between lysine residues.

1. Formalin is the most common. The standard solution is 10% neutral-buffered formalin. It does not harm the structure of the protein greatly, usually antigenicity is not lost.

2. Paraformaldehyde is a purer version of formalin and is often used in its place as a superior fixative. Penetration is faster than formalin or glutaraldehyde. It must be made from a powder no more than 1 week prior to use.

3. Glutaraldehyde causes deformation of alpha-helix structure, so it is usually not a good choice for immunocytochemistry. However, the fixation is non-reversible and it gives the best overall cytoplasmic and nuclear detail. 2.5% buffered glutaraldehyde is a common fixative for electron microscopy. Tissue should be less than 1mm cubed and the fixative not more than 3 months old.

B. Mercurials fix by an unknown mechanism. They contain mercuric chloride. Penetration is relatively poor, and causes tissue hardness. They give excellent nuclear detail and are best used with hemapoietic and reticuloendothelial tissue. Since they contain mercury, they must be disposed of carefully.

1. B-5 fixative mercury pigment and acid formaldehyde hematein are 2 artifact pigments that must be removed before staining.

2. Zenker's fixative is recommended for lymph nodes, spleen, thymus, and bone marrow. It fixes nuclei very well and gives good detail. The mercury must be removed prior to staining or black deposits will result.

C. Alcohols are protein denaturants and are not routinely used for tissues, as they cause hardness and brittleness. Sprays of alcohol can be used to fix blood smears or PAP smears by physicians, but cheap hair sprays do just as well.

1. Methanol

2. Ethanol

D. Oxidizing Agents cross link proteins, but cause extensive denaturation.

1. Potassium permanganate

2. Potassium dichromate

3. Osmium tetroxide fixes fats and lipids well and imparts a black coloration to them.

E. Picrates- unknown method of action. Produces good nuclear detail, but now as much hardness as mercurials.

1. Picric acid is explosive in it's dry form. When in solution it stains everything yellow.

2. Bouin's solution contains picric acid. It is recommended for testis, connective tissue, GI tract, and endocrine tissue.

FACTORS AFFECTING FIXATION

A. Buffering solutions are used to regulate the pH of the fixative. The best fixation is usually carried out at a neutral pH (6-8). Lower pH can cause formalin-heme pigment that will appear as black as polarizable deposits in tissue.

1. Phosphate
2. Bicarbonate
3. Cacodylate
4. Veronal

B. Penetration depends on the diffuse ability of the fixative. The best penetrators are formalin and alcohol. Glutaraldehyde is the worst. No fixative will penetrate more than 2-3mm of solid tissue or 0.5 cm of porous tissue in a 24 hour period.

C. The volume of the fixative is ideally at a 20:1 ratio of fixative to tissue. Agitation and frequent changes of fixative should help to insure good fixation.

D. An increase in temperature will increase the speed of the fixation. Be careful not to "cook" the tissue. Heat will coagulate proteins quickly, but also speeds autolysis.

E. The concentration should be adjusted to the lowest level possible. A high concentration is costly and will introduce artifacts similar to excessive heating. F. The time interval between tissue removal and fixation is critical. Longer periods of time will introduce drying artifacts, lost cellular organelles, nuclear shrinkage, and artifactual clumping. Most tissues should remain in fixative for 24 hours, then stored in 70% ETOH.