The histological processing includes a variety of methods and techniques aimed to study the molecular and morphological features of tissues. Different sets of experimental methods are used depending on what we want to study. However, there are common protocols used in many histology labs for processing tissues to be observed in light or electron microscopes (Figure 1). Methods and techniques can be adapted and combined in many ways to achieve the results that better fit our goals.
Histological processing begins with the collection of tissue samples to be studied. In plant histology, pieces of tissues are removed directly from the plant body, whereas in animal histology there are two options: get a sample of an organ or tissue, a biopsy from the living animal or a necropsy from a dead animal, and then start the histological processing, or start the histological processing with the complete body. In this case, the sample can be removed and further processed at later stages of the histological processing. In any case, samples (or the whole body) are first fixed with solutions known as fixatives. They keep the features of tissular molecules and structures while sample goes on through the successive histological techniques. Fixation is like having a photo of the sample in the living organism, its organization and features are unaltered during the processing, and they are still preserved when the sample is observed with the microscope. Another way to fix tissues is by fast freezing. This type of fixation is used when chemical fixatives or other steps of the processing affect the tissular features we are interested in. For example, some molecules are modified by fixatives or chemical solutions in later steps.
After fixation, the next common step is embedding of the sample in order to obtain sections of the tissue. Embedding is the infiltration of the sample with liquid substances that are later solidified by temperature (paraffin) or by polymerization (resins). The thinner we want the section, the harder should be the embedding medium. Hardening, and therefore thin sections, can be also achieved by freezing the sample. Thicker sections (for example, 40 µm) can be obtained without embedding by using special sectioning devices like vibratome and freezing microtomes. Usually, embedding compounds are not hydrophilic. Thus, first, the water must be replaced with a lipophilic solvent, which in turn is replaced with the embedding substance.
After embedding, or freezing, samples are cut in sections. There are a number of devices for cutting that yield sections with different thickness: ultrathin sections (dozens of nanometers), semithin sections (between 0.5 and 2 micrometers), thin sections (between 3 and 10 micrometers) and thick (thicker than 10 micrometers). Sections are further processed, which usually involves staining with hydrophilic dyes. However, there are several types of microscopy techniques, such as phase contrast microscopy, that allow the visualization of tissular features without section processing. If hydrophilic dyes are used, the embedding media must be replaced with water, otherwise dyes do not enter the tissue. Semithin sections (for light microscopy) and ultrathin sections (for electron microscopy) obtained from resin embedded samples can be stained with dyes or contrasted with heavy metals, respectively, without removing the resin. In sections obtained from frozen samples, dyes are applied once the tissue is thawed.
Processed tissues are studied with microscopes. A variety of types of microscopes area available, but light and electron microscopes are the more common. Light microscopes relay on visible light and glass lens. Several light microscopy set-ups are available: clear field, phase contrast, polarization and differential interference contrast. Electron microscopes give a far larger magnification that allows the study of the so-called ultrastructure of cells and tissues, which include cell membranes and even large molecules.
As we said above, there are many possible modifications of the general histological process. For example, samples can be observed with the scanning electron microscopy without the need for embedding and cutting, but only surfaces can be studied. If surfaces, volumes or lengths of tissular structures have to be quantified, a systematic and random sampling should be designed before obtaining the sections to be sure that all elements have the same probability to be measured. If the structures to be studied are very large and must be visualized in situ in the complete sample, a treatment for tissue clearing must be done. In the following pages, the more common techniques for studying tissues are shown.