At light microscopy, most 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 cells and structures. When the 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 have been used as dyes in both animal and plant histology from the mid-19th century to 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 study 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 techniques, like transgenic animals with modified genes that, when expressed, produce fluorescent proteins like the green fluorescent protein (GFP), that can be observed with fluorescence microscopes. Preparing the tissues for a better staining or labeling has been also highly improved. For example, selecting specific fixatives, permeabilization of thick sections, sophisticated cutting techniques, and many others.
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 electrochemical affinity.
b) Histochemistry includes 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 a host animal 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 is a technique based on the complementary binding of nucleotide (adenine with thymine/uracil, and guanine and cytosine). In this way, two single strands of nucleotides with complementary sequences specifically join together to form a double strand. This process is called hybridization. In situ hybridization is used in histology to study gen expression by detecting a particular messenger RNA (mRNA). A complementary sequence, referred to as probe, is synthesized and labeled with a marker, and used to hybridize with a particular mRNA in the section. In situ hybridization is a highly specific detection technique because only complementary mRNAs are hybridized.