Sample preparation/fixation

Legions of investigators, and many manufacturers, have addressed different aspects ofthe problem, focusing upon better sample preparation (fixation), more effective methods of antigen retrieval, improved reagents, more sensitive detection methods, and the development of reference standards or controls (2-8). To date, these approaches have failed to produce an overall system of IHC that assures uniform high quality, with a level of reproducibility and reliability sufficient to allow robust comparison of IHC results across laboratories, especially where semi-quantitative results are sought (as for Her 2, or ER) (4, 7, 8). 

Resolution of pre-analytic issues (sample preparation, fixation) will require an order of general collaboration hither to unseen, even should there be agreement as to which new and better fixative to use. This author believes that replacement of formalin on a large scale is unlikely in the next decade therefore we must seek to improve reproducibility within that limitation. 

Beyond following detailed guidelines, marked improvement in standardization may also be achieved by the use of reference standards, against which the impact (adverse or otherwise) of sample preparation (fixation) can be measured. This will be discussed in greater detail following the section on validation of reagents, protocols, and staining results. 

Sample preparation for AFM analysis is relatively simple. Generally, a desired amount of sample is absorbed onto a smooth and clean substrate surface, for example, a freshly cleaved mica surface. For example, to prepare a food macromolecule sample for AFM imaging in air, the diluted macromolecule solution is disrupted by vortexing. Then, a small aliquot (tens of microliters) of vortexed solution is deposited onto a surface of freshly cleaved mica sheet by pipette. The mica surface is air dried before the AFM scan. A clean surrounding is required to avoid the interference of dust in the air. Molecular combing or fluid fixation may be applied to manipulate the molecule to get more information. 

Standardization of IHC/ICC has been a critical issue for more than three decades, especially with the advances in targeted therapy such as the development of trastuzumab (Herceptin) for advanced breast cancer.51 Nevertheless, standardization is a difficult issue because numerous factors may influence the consistency and reliability of immunostaining results, including fixatives, fixation time, AR, antibody clones, detection system, and interpretation (see Part II). In cytopathology, the situation is even worse due to its variable cell sample preparation techniques. Cytopreparation is. .. the foundation of cytomorphology. 52 We believe it is also the foundation of ICC. Therefore, standardization of ICC needs to start with uniform and reliable cytopreparation. 

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. 

There are a number of techniques that are used to elucidate the structure of tissues at the light and electron microscopic levels. At the light level different techniques are used to process tissue for making standard paraffin sections that are the gold standard of light microscopic sample preparation. The steps used to prepare paraffin sections include fixation,... 

The net adsorption enthalpies and the predicted sublimation enthalpies (see Method 1) were used to calculate the adsorption enthalpies of transactinides on selected metal surfaces (Method 11). The metals, which are presented in Table 4, can be used as stationary phase in gas adsorption chromatographic experiments for selective gas chemical separations or, in the case of high adsorption interaction, as strong fixation materials for the sample preparation in the measurement of transactinides. 

The usual method of sample preparation for tissue remains as formalin fixation and paraffin embedment (FFPE). This venerable approach may be satisfactory for the preservation of morphologic detail, but does adversely affect the antigenicity of many target molecules in the tissue, to degrees that are unknown. The enormous variation in protocols (including fixation times) employed for FFPE among different laboratories, or within the same laboratory from specimen to specimen, compounds the problem, and contributes to the current poor reproducibility. 

Tissue Processing Skinned mouse knee joints are immersed in neutral buffered formaldehyde (3.7 %) directly after preparation. Fixation times are limited to one week to ensure tissue integrity. Tissue samples are rinsed overnight with tap water. Tissue samples that contain bone are decalcified (D-calciher normal, Shandon 1779) for 2 or 3 days. Decalcified samples or cartilage samples without bone are rinsed overnight with tap water. After dehydration with alcohol in stepwise increasing concentrations, tissue samples are embedded in paraffin. 

Currently developed for many applications (Stoeckli et al. 1999 Stoeckli et al. 2001, 2002,2003 Chaurand et al. 2004), MALDI MSI is achieved by rastering sequentially the surface of a defined area while acquiring a mass spectrum from every location (see Figure 2). Atypical sample preparation for MSI involves the fixation of the sample, for example, tissue section, on a MALDI plate and the application of the matrix solution over the latter, either as a thin layer or as a spot pattern, to get co-crystallization of analytes with matrix while solvents evaporate. Once dried, the sample is introduced in the mass spectrometer, where, for each defined image position, short UV laser pulses are fired onto the surface to generate ions. Those are analyzed by the TOF instrument and a mass spectmm is acquired. 

Depending on the concentration of DNA used in the sample preparation procedure, the surface coverage can be adjusted. The fixation using the silanization procedure is beneficial in effectively immobilizing the DAN for AFM work. Stability in excess of 5 h has been reported [106] (Fig. 3.50). 

There is the potential to establish internal reference standards that will be of use in assessing the effectiveness of sample preparation and fixation, giving an indication of the suitability of the tissue for IHC studies we will address this new area following the discussion of reagent and protocol validation and at the end of this chapter. 

In vitro staining is used to colour cells or structures that have been removed from their biological context. Certain stains are often combined to reveal more details and features than a single stain alone can provide. Combined with specific protocols for fixation, sample preparation and investigative methods such as fluorescence microscopy, these standard staining techniques are used as consistent, repeatable diagnostic tools. 

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