Histological Stains

Tissue Processing To some extent, light-level (as opposed to e.m.-

level) histology is a lost art, especially if one is interested in producing research-quality histological sections and photomicrographs. Thus, practical resource materials are somewhat limited. Although I will list the resourses that we use, if you are just beginning I strongly encourage you to learn fixation, embedding, sectioning, etc. from a good histologist, and preferably one that has worked at the light level.

Tissue Processing A good general reference that we use routinely

is the Manual of Histological Methods by Prophet et al. (1992), and also the earlier version of this manual (Luna 1968), which is produced by the Armed Forces Institute of Pathology. This manual contains very general discriptions of tissue processing, as well as a variety of general staining and counterstaining methods that are quite useful.

Tissue Processing

 a) Fixation        i) Standard Fixation        ii) Cryopreservation    

b) Embedding        i) Clearing        ii) Infiltration        iii) Orientation    

c) Sectioning (microtomy)        i) Microtone         ii) Cryostat

Fixation

Fixation is one of the most critical aspects of histology, because it determines a large extent which tissue components you will be able to detect. For example, I have heard the claim that cryopreservation and cryosectioning are the methods of choice for light level immunohistochemistry because antigens are preserved so well. We have seldom used this method because they give mediocre tissue morphology. We have had good success detecting a host of molecules immunohistochemically using fixed paraffin-embedded tissues, which gives good tissue preservation.

Fixation

I believe a key to our success has been that we routinely use several different fixatives when we collect tissues. For example, we routinely fix samples of a given tissue in formalin and also Carnoy's along with cryopreserving a sample. Another method used has been Bouin's. We invariably find that one-fixation method is preferable to the others for detection of a given antigen in a given tissue.

Staining reactionsThe mechanisms involved in staining include the following:

The dye may actually be dissolved in the stained substance.

Most fat staining is accomplished in this fashion. A dye may be absorbed on the surface of a

structure, or dyes may be precipitated within the structure, simply because environmental factors (pH, ionic strength, temperature, etc.) favor absorption or precipitation.

Most staining reactions involve a chemical union between dye and stained substance through salt linkages, hydrogen bonds, or others.

Staining with these dyes results in a predictable color pattern based in part on the acid base characteristics of the tissue.

Color and color distribution are not absolutely reliable for discrimination between tissue components.

Color will vary not only with specific stains used, but also with the conditions that exist during preparation of the slide.

These include everything from the initial fixing solution to the ionic strength of the staining solution and the differentiating solvents utilized after staining.

Acid and Basic Dyes

Most histologic dyes are classified either as acid or as basic dyes. An acid dye exists as an anion (negatively

charged) in solution, while a basic dye exists as a cation (positive charge).

The hematoxylin-eosin stain (H&E), The hematoxylin-metal complex acts as a basic

dye. The eosin acts as an acid dye.

Acid and Basic Dyes

Any substance that is stained by the basic dye is considered to be basophilic; it carries acid groups which bind the basic dye through salt linkages. When using hematoxylin, basophilic structures in

the tissue appear blue (or purple or brown; this varies according to the stain that is being used).

A substance that is stained by an acid dye is referred to as acidophilic; it carries basic groups which bind the acid dye. With eosin, acidophilic structures appear in various shades of pink. Since eosin is a widely used acid dye, acidophilic

substances are frequently referred to as eosinophilic.

H&E stain

H&E is without doubt the most common stain used in histology.

Well over 90% of the slides you'll look at in your life will be stained with it. It's actually a combination of two dyes: the basic dye hematoxylin, and the alcohol-based synthetic material, eosin.

H&E is a structural stain, primarily giving you morphological information.

E&H Stains

In an H&E stain you'll usually see both eosinophilia and basophilia: the nuclei of cells basophilic, while eosinophilia is typical of cytoplasmic constituents.

The appearance of a tissue in H&E is what we've come to regard as it's "actual" or "normal" or "real" one, and we use it as a basis for comparison when special stains are applied to reveal some other aspect of the tissue's structure or chemistry.

E&H Stains

The staining reaction is clearly stronger in some parts of the tissue and cells than in others, allowing identification of the details

Heart

Kidney