Fixation involves submerging the sampled tissue in chemical substances (i.e. fixatives) in order to prevent tissue digestion by enzymes or bacteria and to preserve as much as possible of its morphologic and chemical characteristics. Fixatives promote cross-links between proteins and form a gel that maintains the in vivo relations of tissue components to each other [14]. There are a number of reagents that can be used for fixation, each of which has differing penetration rates. Formaldehyde [15] is the most commonly used agent for histopathology and when dissolved in water, it is referred to as “formalin.” One part formalin is typically diluted with nine parts water to produce a 10 % formalin solution, a concentration that is optimal for tissue fixation [16]. This solution penetrates tissue at about 1 mm an hour [17], therefore, biopsies are generally submitted for processing the same day as received, while larger specimens (e.g. a mastectomy) are not processed the same day as received as they require a longer fixation period.

The processing of tissue includes dehydration, clearing, and embedding steps. Dehydration involves the removal of fixative and water from the tissue and their replacement with dehydrating agents by placement in increasing concentrations of ethanol. Next, the clearing step involves replacing the dehydrating fluid with a lipid solvent (e.g. xylene). The tissue is subsequently removed from the cassette and placed in a molten wax-filled mold for embedding. At this point, orientation of the tissue within the mold is critical, as it will determine the plane through which the section will be cut. Incorrect placement of tissues may result in diagnostically important areas being missed or damaged later [18]. Elongate tissues should be placed diagonally across the block (e.g. core biopsy), while tubular structures (e.g. vas deferens) are embedded so as to provide transverse sections showing all tissue layers. Specimens that have an epithelial surface (e.g. skin surface) are embedded in such a way as to provide sections in a plane at right angles to the surface.

The waxed cassette is next placed in an instrument with fine blades called a microtome. Rotation of the drive wheel moves the block holder a controlled distance forwards, the blade’s edge strikes the tissue block, and thin sections are cut and affixed to a glass slide. Sections are usually four microns thick so that a single layer of cells can later be seen under the microscope. Following thorough drying of the tissue sections are ready for staining.

IHC is a multistep technique involving the interaction of a target antigen (i.e. the protein of interest) with a specific antibody tagged with a visible label [24]. The aim is to detect the presence of elevated levels or the absence of a particular target antigen. Antibodies can be coupled with an enzyme, such as horseradish peroxidase (HRP), requiring the use of a light microscope for visualization, or with fluorescent chemical compounds requiring the use of a fluorescent microscope. Different labeling techniques are available including the direct or indirect labeling method. In a direct assay, a fluorophore-labeled antibody, such as fluorescein isothiocyanate, can react directly with the target antigen. This tag allows immediate visualization of the antigen. In contrast, in an indirect assay, an unlabeled primary antibody is used. This binds to the target antigen and an enzyme-labeled secondary antibody binds to the primary antibody. In general, primary antibodies are raised against the antigen of interest and are unlabeled, while secondary antibodies are raised against IgG of the primary antibody. Finally, a chromogenic substrate must be added for visualization (e.g. diaminobenzidine), which reacts to produce a brown precipitate in the presence of the HRP enzyme. A simplistic illustration of these two assays is shown in Fig. 2.1. Recent advances in this area include multiplexing of antigens [25, 26], whereby multiple stains can be performed on the same tissue section, followed by analysis using digital imaging software.

While IHC involves the detection of marker proteins in tissue, ISH can detect target ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) sequences [27]. Different detection systems are then used to visualize the presence of the target sequence. Fluorescence ISH (FISH) uses fluorescent dyes and fluorescent microscopy while chromogenic ISH (CISH) uses chromogenic dyes and brightfield microscopy [28]. In this process, a complementary DNA strand (or probe), or RNA strand (or riboprobe) is used to localize a specific DNA or RNA sequence. The probe hybridizes to the target sequence with elevated temperature (i.e. denaturation) and excess probe is washed away. If the probe is already fluorescent, it will detect the site of hybridization directly. If the probe is chromogenic, an additional step is needed to visualize the probe [29]. A simplistic illustration of ISH is shown in Fig. 2.2. Multiplexing using ISH can also be performed [30], followed by spectral imaging for the detection and subsequent deconvolution of multiple signals.

The cytoplasm surrounds the cell nucleus and is surrounded by a plasma membrane, which separates the interior of the cell from the outside environment. This membrane is selectively permeable and controls the movement of substances in and out of the cell. The cytoplasm is made largely of cytosol fluid containing multiple organelles (“little organs”) suspended within it that carry out specific directions of the nucleus. These organelles include mitochondria, endoplasmic reticula, the Golgi apparatus, vacuoles, and lysosomes (Fig. 2.5) among others. Mitochondria function to produce most of the cell’s energy in the form of adenosine triphosphate (ATP) [42] and are also involved in processes such as cell signaling [43], cellular differentiation [44], cell growth [45], cell cycle control [46], and cell death [47]. There are two types of endoplasmic reticula, the rough endoplasmic reticulum (RER) and the smooth endoplasmic reticulum (SER) [48]. The SER is involved in lipid metabolism [49], carbohydrate metabolism, and detoxification, while the RER contains ribosomes on the surface where active protein synthesis occurs [50, 51]. The Golgi apparatus concentrates and packages proteins from the RER inside the cytoplasm prior to being transferred to their appropriate destinations [52]. Vacuoles are involved in the storage and intracellular digestion of molecules and lysosomes contain enzymes responsible for the breakdown of proteins, nucleic acid, carbohydrates, lipids, cellular debris, and foreign organisms [53].

The nucleus is the largest organelle found in human cells and is surrounded by a nuclear envelope with nuclear pore complexes that allow material to move in and out [54]. It contains genetic information in the form of DNA. DNA is a complex molecule consisting of two antiparallel strands of nucleotide bases, each with a backbone of sugar (deoxyribose) molecules linked together by phosphate groups [55]. Each sugar molecule is linked to a base, which is attached by hydrogen bonds to a base on the other strand in a complementary fashion so that adenine (A) bonds with thymine (T) and guanine (G) bonds with cytosine (C) [56]. DNA is organized into highly compact, regular units called chromosomes. Within the nucleus is a structure called the nucleolus, which contains RNA, ribosomal proteins, and functions as the site of ribosome synthesis. RNA differs from DNA in that RNA molecules are single-stranded, the backbone sugar is ribose, and it contains uracil (U) in place of thymine [57].

Human genetic testing has made significant advances in the past decade [58]. The identification of certain sequences in order to diagnose genetic diseases can be done by performing sequencing on a blood sample, fresh tissue, or paraffin embedded tissue. DNA sequencing [59] is the process of determining the nucleotide order of DNA fragment. As mRNA is generated by transcription from DNA, reverse transcription must be performed (using a reverse transcriptase enzyme) for RNA sequencing [60]. Following this, complementary DNA (cDNA) fragments are generated, PCR amplification executed, a library created, and sequencing performed comparing results to a reference genome.

Epithelial cells are generally classified as “covering and lining epithelia” which are found lining the cavities of the body and surfaces of structures, or as “glandular (or secretory) epithelia” [41]. Glands refer to single cells or groups of cells that secrete protein, mucus, or lipid. This includes “endocrine glands” which secrete into extracellular spaces (i.e. secrete internally) and “exocrine glands” which secrete into ducts (i.e. secrete into the external environment). Epithelial cells can also be classified based on the number of cell layers present and the shape of the cells in the top layer (Fig. 2.6). Epithelial tissue can, therefore, be one cell thick (i.e. simple epithelium), or two or more cells thick (i.e. stratified epithelium) [61]. There are three basic cell shapes based on microscopic appearance, including squamous (flat and wide), columnar (tall), and cuboidal (cube shaped) cells. Consequently, by describing the number of cell layers and the surface cell shape the different forms of epithelia can easily be classified. In some cases a third feature, namely specialization of the cell surface, e.g., keratinization [62] or the presence of cilia [63], is included. Stratified squamous epithelium that is exposed directly to the environment (e.g. the skin), can show keratinization (i.e. a layer of dead cells is present on the surface), while those that are not directly exposed (e.g. the oral cavity) are only partially keratinized or nonkeratinized. The presence of cilia (i.e. hair-like motile processes) is another specialization seen in simple columnar epithelium present on the surface of these cells. This surface adaptation helps propel substances along, e.g., the airways contain cilia to propel mucus.