At light microscopy, most of the animal tissues are colorless, excepting those containing some pigments like hemoglobin in the blood or melanin in the epidermis. However, plant tissues show a wider variety of pigments that allow some light microscopy studies. The cell wall helps to discern plant tissue cells and structures. When first light microscopes were invented, lab researches had to discover how to stain tissues in order to observe their morphological features. Some common pigments like carmine and eosin, dissolved in water, could stain some tissue structures. The expansion of the fabric industry in the XIX century, and the need to color the clothes, led to a fast development of a large diversity of dyes and pigments. Many of these substances were used as dyes in both animal and plant histology from the mid-19th century nowadays. During this time, histological staining has witnessed an enormous development with new techniques and synthetic dyes that fulfill most of the researcher needs. Molecular biologists have developed new techniques to label cells and tissues, such as immunostaining by using antibodies, or in situ hybridization by using ARN and DNA labeled probes. There are even more sophisticated techinques, like transgenic animals with modified genes that, when expressed, produce fluorescent proteins like the green flourescent protein (GFP), that can be observed with fluorescence microscopes.
In these pages, we are going to deal with basic and common techniques used in most histology labs, but we won't go into detailed protocols of more complex techniques. We divide the common hitological techniques in five groups:
a) General staining are staining techniques that use colored substances, or dyes, that binds to tissular structures by electro-chemical affinity.
b) Histochemistry include those techniques involving chemical modification of some molecules already present in the tissues that allow the binding of dyes. In this section, staining methods based on the enzymatic activity of tissular enzymes will also be included.
c) Lectins, such as selectins, are proteins bearing molecular domains that are able to recognize carbohydrates and carbohydrate bonds. The recognition is so specific that lectins are used to identify carbohydrates present in glycoproteins of cell membranes and extracellular matrix, as well as mucopolysaccharides. Lectins recognize some cell types and tissues, and are used as tools to study those tissular components.
d) Immunohistochemistry is a very powerful technique based on the specificity of the antibodies. Antibodies are produced as a result of the immune response of an animal host after the injection of a foreign molecule, the antigen. Purified antibodies from the host animal are used to recognize this molecule in tissue sections.
e) In situ hybridization are used in histology to study gen expression by detecting messenger RNA (mRNA). It is based in the recognition of two nucleotide single strands having complementary sequences. This recognition is known as hybridization. One of the sequences is called probe, which is labeled with a marker and used to identify a particular mRNA in the section. In situ hybridization is a highly specific detection technique because only complementary mRNAs are hybridized.