Fixation, sample tissue preparation

Prevention of non-immunological adsorption of immuno-reactants to tissue preparations. The frequent use of cross-linkers both in the preparation of conjugates and fixation of the samples may result in a stickiness of the preparations. Most of these crosslinkers react via amino groups. Molin et al. (1978) reduced background staining by the inclusion of 20 mM lysine, with or without 1% BSA, in the incubation steps. 

Those interested in using immunohistochemistry to study apoptosis need to consider the method of tissue preparation and fixation. Since many antibody epitopes do not survive formalin/glutaraldehyde fixation or paraffin embedding, investigators should determine under what conditions the antibody of interest will work prior to sample collection. There are antibodies that will successfully bind to formalin-fixed, paraffin-embedded material, but if the investigator is unsure, fresh snap-frozen samples can be used to optimize conditions for success since freezing generally will not alter epitopes. 

Tissue preparation consists of fixation, subsequent dehydration, and embedding in paraffin wax to provide a rigid matrix for sectioning. Tissues that are to be embedded in paraffin wax are first fixed to optimize preservation, a process that profoundly affects the morphologic and immunohistologic results. The ideal fixative for IHC studies should not only be readily available but also be in widespread use to maximize the range and number of samples available for... 

This chapter summarizes our experience with this approach and describes some of the critical parameters involved in the preparation of rat neural tissue for LCM, with particular emphasis on protocols applied to the Arcturus Autopix LCM. This instrument has been superceded by more recent models, which are now marketed and distributed by MDS Analytical Technologies (http // www.moleculardevices.com). The principles described here, however, are applicable to tissue preparation for most LCM instruments. We describe two basic protocols for the isolation of samples of rat brain, first from unstained microdissected regions, and second, from brain cells that express specific antigens identified by immunostaining. In addition, we compare the effects of different fixation conditions on tissue recovery and RNA content using real-time QPCR. 

The protocols described below illustrate (1) frozen section sample preparation, (2) hematoxylin and eosin (H E) tissue staining, and (3) automated LCM. Alternative tissue preparation methods, such as ethanol or formalin fixation with paraffin embedding, are acceptable for RNA and DNA analysis... 

This chapter contains two main parts, the first part (section 5.2) deals with sample preparation for tissues, and the second part (section 5.3) deals with sample preparation for cells. In section 5.2, tissue preparation methods for spectroscopic analysis such as cryopreservation (section 5.2.2), fixation (section 5.2.3) and embedding (section 5.2.4) are described and a number of studies investigating the... 

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. 

After primary fixation the algal and root samples were rinsed in 0.1 M PIPES buffer, pH 7.0, then twice in 0.1 M sodium cacodylate buffer, pH 6.8, and post-fixed in 1% osmium tetroxide in 0.1 M cacodylate buffer, pH 6.8, overnight at 4°C. The osmium solution prepared for the root samples was added with 0.7% potassium ferricyanide in order to improve osmium penetration in the root tissues. This was particularly necessary for the 2 h control roots and roots from RO-treated seed, due to their relatively low water content. The replacement of PIPES buffer with cacodylate buffer before osmication was necessary, as PIPES reacts with osmium, producing a dark precipitate. 

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 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. 

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. 

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