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Following injury, vascular damage results in the loss of perfusion and consequent low oxygen tension (hypoxia) which may be exacerbated by a rapid influx of inflammatory and mesenchymal cells with high metabolic demands for oxygen. Changes in systemic and cellular oxygen concentrations induce tightly regulated response pathways that attempt to restore oxygen supply to cells and modulate cell function in hypoxic conditions. Most of these responses occur through the induction of the transcription factor hypoxia-inducible factor-1 (HIF-1) which regulates many processes needed for tissue repair during ischemia in the damaged tissue. HIF-1 transcriptionally upregulates expression of metabolic proteins (GLUT-1), adhesion proteins (integrins), soluble growth factors (TGF-β and VEGF), and extracellular matrix components (type I collagen and fibronectin), which enhance the repair process. For these reasons, HIF-1 is viewed as a positive regulator of wound healing and a potential regulator of organ repair and tissue fibrosis. Understanding the complex role of hypoxia in the loss of function in scarring tissues and biology of chronic wound, and organ repair will aid in the development of pharmaceutical agents that can redress the detrimental outcomes often seen in repair and scarring., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Apoptosis is an important process both in normal biology and in various pathologies and disease states. Apoptosis in tissue or cells can be detected in a number of ways. In tissue sections, electron microscopy can identify apoptosis by cellular and nuclear morphology, and in live cells, changes in the membrane and membrane permeability allow apoptosis and necrosis to be observed. Histologically, apoptosis is best detected using the partial DNA degradation that is present in apoptotic cell nuclei. Terminal transferase-mediated UTP nick end-labeling (TUNEL) has been used successfully for detection of DNA degradation in paraffin-embedded tissue sections and can be combined with immunohistochemistry if desired to allow more precise identification of apoptotic cells.

The basics of in situ hybridization have been widely applied to a diverse range of situations where we need to localize the distribution of nucleic acids. Advances in other molecular techniques such as the advent of gene microarrays has not diminished the significance of in situ hybridization, but rather highlight the importance of being able to identify the topology of gene expression. In situ hybridization offers a degree of precision that is unavailable with other molecular techniques. This chapter outlines techniques used to examine the spatial distribution of gene expression in the kidney using complementary RNA (cRNA) probes with both radioactive and non-radioactive labels.

In this chapter we describe the use of cRNA (riboprobes) in the detection of gene expression in tissue sections. Riboprobes offer good sensitivity and allow the detection of low-level mRNA expression. In some cases, the use of radiolabeling is justified because this method is still sensitive. However, recent advances in nonisotopic detection methods mean that in some cases digoxigenin (DIG) or biotin labeling also may be sufficiently sensitive to detect mRNA expression in tissues of interest. The use of alkaline phosphatase conjugated anti-DIG antibodies improves the sensitivity of DIG detection over peroxidase systems, and the use of amplification systems based on biotinyl tyramide has improved the sensitivity of biotin labelled probe detection. Finally, it can be shown that low-level mRNA expression is easier to detect in frozen sections than in paraffin-embedded material, with a consequent loss in quality of morphology.

Cell death by apoptosis is now recognized widely as an important constituent of cell turnover and disease pathology. Characterized by the cleavage of DNA into oligonucleosome-sized DNA fragments, end-labeling of fragmented DNA often is used as an in situ histological marker of apoptosis. The judicious and appropriate use of this technique therefore provides us with an important tool for assessing cell kinetics. Protocols for both terminal transferase-mediated UTP nick end-labeling, so-called TUNEL, and the combination of TUNEL with immunohistochemical staining are presented here, along with a discussion of its significance and interpretation.

Monoclonal antibodies to proliferation associated antigens have long been used to histologically localize mitogenesis. However, techniques that distinguish cells in the synthetic or S phase have tended to rely on the in vivo incorporation of tritiated thymidine or thymidine analogs such as bromodeoxyuridine. The necessity to pulse with these labels before retrieving tissue means that they cannot be used in humans and are not available retrospectively. Measuring expression of histones serves as a useful adjunct to these techniques. As expression of histone proteins (H2A, H2B, H3, H4) are restricted to the synthetic phase of the cell cycle, hybridization for histone mRNA precisely distinguishes those cells in the S phase. Measuring their expression can easily be applied to the histological localization of proliferation, and can be used both prospectively and with archived tissue specimens. Several histone in situ hybridization probes and nonradioactive detection systems are now available commercially. A generalized protocol for their use in measuring in situ proliferation is provided in this chapter.