Tissue fixation techniques

In vivo receptor labeling offers binding conditions similar to those prevailing in the physiological state and it provides anatomical resolutions compatible with perfusion fixation of target tissue. This technique is... 

Improper fixation and processing Proper antigen fixation is the cornerstone of immunoperoxidase techniques, for without it, results will be poor. Some markers may be destroyed by certain fixatives overfixation may mask certain antigens (see Chapter 8) (4). Review manufacturer s package inserts for appropriate fixation techniques. Paraffin-embedded tissue should never be exposed to temperatures >60°C as this can destroy some antigens. 

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. 

Comparisons have been made of HA localization in skin sections fixed with acid-formalin/ethanol and conventional formalin fixation. Much of the HA, particularly in the epidermis, is eluted during the process of formalin fixation. This suggests that epidermal HA is more loosely associated with cell and tissue structures than is dermal HA. A further incubation of 24 h in aqueous buffer further increases the disparity between the acid-formalin/alcohol and the conventional fixation technique. Once the tissue has been exposed to the acid-formalin/alcohol, the HA association with tissue becomes tenaciously fixed, with little loss of apparent HA observed following additional aqueous incubation, while the formalin-fixed tissues demonstrate progressive loss of HA. 

Tissue fixation The techniques used to evaluate tissue structure (e.g., light microscopy, confocal microscopy, or electron microscopy), will vary with the goals of the experiment. Proper tissue processing and fixation should focus on preserving tissue structure and tissue antigens. 

Tissue Fixation, Processing, and Antigen-Retrieval Techniques 18... 

TISSUE FIXATION, PROCESSING, AND ANTIGEN-RETRIEVAL TECHNIQUES... 

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... 

Vollmer E, Muller AM, Muller-Navia J (2001) HOPE fixation a novel fixing method and paraffin-embedding technique for human soft tissues. Pathol Res Pract 197 823-826 7. Werner M, Chott A, Fabiano A, Battifora H (2000) Effect of formalin tissue fixation and... 

Comparison of Fixation Techniques. For microwave fixation, fresh tissues were trimmed to approximately 2 mm thickness, placed in plastic cassettes, and held in ice cold saline (30-120 minutes) until irradiation in a 700-watt oven set to hold at 60°C for 2 minutes (7). Microwave-fixed tissues were stored overnight in 70% ethanol at room temperature, processed routinely and then embedded in paraffin, sectioned, deparaffinized, and rehydrated using standard techniques. Other pieces of liver were similarly trimmed, then fixed by immersion in either 3.7% formaldehyde in neutral phosphate buffer (48 hours), 2% glutaraldehyde in phosphate buffer (2 hours), Bouin s solution (24 hours), or B-5 solution (4 hours). These tissues were similarly embedded in paraffin then sectioned routinely. In other studies, specimens of lung and kidney were fixed by microwave irradiation with the same schedule as liver. 

S. Knecht, C. Erggelet, M. Endres, M. Sittinger, C. Kaps, E. Smssi, Mechanical testing of fixation techniques for scaffold-based tissue engineering grafts, J. Biomed. Mater. Res. B Appl. Biomater. 83 (2007) 50-57. 

There are basically only two protocols that we use for in situ hybridization to whole-mount tissues, and in essence they are identical one is for whole-mount specimens that can be handled in Eppendorf tubes and the other is adapted for whole-mount specimens on slides. As described above, the challenge in extending these methods to new tissues is to find isolation and fixation techniques that enable them to go through one of these procedures. 

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