Immunohistochemistry tissue processing

Figure 5.3 Diagram depicts the further-designed studies to test our hypothesis with respect to standardization of immunohistochemistry based on the antigen retrieval technique exemplified in a multiple direction to draw a conclusion, (a) Periods of formalin fixation, (b) Variable delay of fixation, (c) Storage of FFPE tissue blocks or sections, (d) Variable thickness of FFPE tissue sections, (e) Other variable conditions of processing FFPE tissue blocks. The stereoscopic frame of a cube represents the reliable limitation of quantitative IFIC demonstrated by serial studies as recommended in the text. Reproduced with permission from Shi et al., J. Histochem. Cytochem. 2007 55 105-109.

Wester K, Andersson A, Ranefall P, et al. Cultured human fibroblasts in agarose gel as a multi-functional control for immunohistochemistry. Standardisation of Ki67 (MIB1) assessment in routinely processed urinary bladder carcinoma tissue. J. Pathol. 2000 190 503-511. 

Eltoum, I., Fredenburgh, J., and Grizzle, W. E. 2001. Advanced concepts in fixation. 1. Effects of fixation on immunohistochemistry, reversibility of fixation and recovery of proteins, nucleic acids, and other molecules from fixed and processed tissues. 2. Developmental methods of fixation. J. Histotechnol. 24 201-210. 

Shi, S.-R., Gu, J., Kalra, K. L., Chen, T., Cote, R. J., and Taylor, C. R. 1995b. Antigen retrieval technique A novel approach to immunohistochemistry on routinely processed tissue sections. Cell Vision 2 6-22. 

Werner, M., Chott, A., Fabiano, A., and Battifora, H. 2000. Effect of formalin tissue fixation and processing on immunohistochemistry. Am. J. Surg. Pathol. 24 1016-1019. 

Raman spectroscopy can offer a number of advantages over traditional cell or tissue analysis techniques used in the field of TE (Table 18.1). Commonly used analytical techniques in TE include the determination of a specific enzyme activity (e.g. lactate dehydrogenase, alkaline phosphatase), the expression of genes (e.g. real-time reverse transcriptase polymerase chain reaction) or proteins (e.g. immunohistochemistry, immunocytochemistry, flow cytometry) relevant to cell behaviour and tissue formation. These techniques require invasive processing steps (enzyme treatment, chemical fixation and/or the use of colorimetric or fluorescent labels) which consequently render these techniques unsuitable for studying live cell culture systems in vitro. Raman spectroscopy can, however, be performed directly on cells/tissue constructs without labels, contrast agents or other sample preparation techniques. 

Fig. 4 Immunohistochemistry results for cell-free CCP and TE-CCP scaffolds at 4 and 8 weeks, (a) Staining for the immunomodulatory and tissue remodeling (M2 phenotype) marker CD163 at 4 weeks demonstrated active M2 macrophage activity in the scaffold substance, particularly at sites of new bone formation and the host-scaffold interface in cell-free scaffolds. These cells were less evident by 8 weeks, suggesting an advanced stage of the remodeling process, (b) In the TE scaffolds, M2 phenotype macrophages were evident at both 4 and 8 weeks predominantly at the periphery of the scaffold, especially in the fibrous/inflammatory capsule seen previously on histological examination, (c) The proinflammatoiy (CCR7) marker demonstrated little Ml macrophage cell activity in unseeded scaffolds at either 4 or 8 weeks, (d) There was marked population of these cells at the periphery of TE scaffolds at both 4 and 8 weeks. Reproduced with permission from Lyons et al. [86]...

The perceived need to identify objective markers to supplement, or conceivably supplant, the more subjective established histologic parameters has been a major driving force behind biomarker discovery efforts. It is crucial to recognize and account for the potential variability that can exist even with the new molecular parameters. Sources of variability include differences in molecular technique methodologies, tissue fixation and processing, interobserver and intraobserver variability (in immunohistochemistry-based biomarkers), and differences in cutoff points. Furthermore, illustration of statistical significance for a particular biomarker does... 

One of the areas in which the use of immunocytochemistry has had the greatest impact is in the examination of tissue in the medical pathology laboratory. Immunocytochemistry, or actually, immunohistochemistry, in the pathology laboratory, enhances the study of diseased tissue. It is important, when studying disease, to obtain and process the tissue as quickly as possible. The reason for this is so that the cellular constituents can be preserved as completely as possible. As is the case with any solid piece of... 

Human tissue should be fixed in 10% neutral buffered formalin and then dehydrated for embedding in paraffin. Paraffin is nonaqueous embedding medium, so the tissue blocks must have the water removed or be dehydrated. Dehydration is done in organic solvents such as alcohol, acetone, xylene, or toluene. After dehydration, the tissue blocks are embedded with liquid (warm) paraffin. When cooled, the wax embedded block is sectioned on a rotary microtome. Before immunocytochemistry can be performed on the resulting tissue sections, they must be rehydrated by processing with the same organic solvents back to water. Thus, the dehydration and rehydration steps are needed before immunohistochemistry. 

Performing immunohistochemistry on these rehydrated paraffin sections frequently leads to poor results. In contrast, the same antibodies on paraformaldehyde-fixed and cryostat sections will give good results. The issue with formalin-fixed and paraffin-embedded tissue is that the exposure to formalin and dehydration alters the epitopes in the tissue. As a result, formalin-fixed and paraffin-embedded tissues need additional processing methods, known as epitope retrieval or antigen retrieval. Done before immunohistochemistry, epitope retrieval involves heating the sections in buffer with either an acid or base to allow the antibody to recognize the epitope. Also, the exact process of epitope retrieval can be different for individual antibodies. There are numerous papers and books on epitope retrieval and how to apply the method. 

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