Subject tissue samples

The in situ RT-PCR process subjects tissue samples to various severe chemical and enzymatic reactions and temperature fluctuations. For that reason, silanated or positively charged slides must be used that are able to retain the tissue sample throughout the process. 

The effect of heating on aqueous solutions of ivermectin is difficult to study due to the very low solubility of the compound in water. Thus, the stability of ivermectin has been only investigated on incurred tissue samples. When a series of incurred pig and cattle muscle and liver samples and salmon muscle samples were subjected to various cooking processes, some loss of ivermectin was observed but the loss was associated with the leakage of the fat from the tissue samples... 

Semisolid samples such as muscle and liver tissues can be homogenized by blending with water or an appropriate aqueous solution such as a buffer in a mechanical or an ultrasonic device to expose the residue to the extraction solvent. Fatty tissue samples are sometimes subjected to heating at 40 or 60 C until fat becomes liquid, prior to extraction of the analytes with hexane (433) or acetonitrile (434), respectively. An alternative pretreatment approach is the enzymatic digestion of the tissue by means of proteolytic enzymes such as subtilisin A (429, 435-437). 

Conventionally, the first attribute known about an enzyme used to be its function, usually in a crude extract. This property was screened for in microbial cultures or in tissue samples. The crude extract was then purified to homogeneity and the protein subjected to biochemical studies to learn of its pH and T profiles, its pi and subunit composition, catalytically important residues, and other properties. Proteolytic digestion of the protein with subsequent Edman degradation led to the primary sequence, but no information on the secondary structures such as a-heli-ces and [5-sheets or the folding in three dimensions of the polypeptide chain. The primary sequence could have been used to deduct the gene sequence but, with the degeneration of the code, several possibilities for certain amino acids occur, which makes prediction of the gene sequence a risk. 

Microtissue arrays are a possible solution to the limited supply of control tissue. Microarray blocks allow the incorporation of 200-300 fine tissue cores into blocks that can be used for controls against a wide variety of antibodies and as the cores are small (0.5-1.5 mm diameter) much of the original tissue block remains preserved. Microtissue arrays should be used with the recognition that each core of tissue has been subjected to different fixation and processing so that the level of antigen preservation in each of the 200-300 tissue samples are different and by no means standardized. 

Another method of validation is the use of standard samples from an approved source but such sources and tissue are not currently available. The use of consensus positive and negative tissue in the form of tissue microarrays is a possible substitute (Fitzgibbons et al, 2006). Alternatively, tissue samples from cases accessioned by your own laboratory known to harbor the target protein by non-IHC means can be used, but in all these situations it has to be remembered that there is no fixation or processing standard so that agreement between laboratories and between samples is subject to pre-analytical variables discussed previously. Clearly the ideal validation procedure would be against patient outcome but this is a costly exercise and often not practical as they require appropriate numbers of patients and a prospective study. 

The use of the avidin-biotin system enables a separation of the primary and secondary steps. Previous employment of an antibody-marker for direct detection was problematic since such conjugates are very large and subject to a variety of irrelevant interactions that interfere with the desired immunochemical binding. In contrast, the biotinylated antibody can first be incubated with the cell or tissue sample following this initial interaction, the sample can be fixed and incubated subsequendy with... 

The retinoid toxicology of fish is a new subject, with only a decade s worth of research. The reduction of retinoid stores with toxicant exposure has been well documented however, the mechanism of this reduction and the implications on RA and fish physiology and health have not been determined. This is partially due to the lack of technology that would allow the easy measurement of RA in fish tissues (required in some remarkably small tissue samples compared to mammals ). It is very important for future studies to establish whether reductions of stored retinoids alter RA levels and whether changes in RA are responsible for the effects of a toxicant observed in fish. However, this may be a challenge in that long-term studies (months to years) may be required to induce retinoid deficiency in fish. 

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