Tissue preparation protocols

Immunocytochemical methods have been widely applied to visualize proteins, carbohydrates, or lipids in sectioned material. The advantage of using immunocytochemistry is to be able to localize the molecules of interest within the tissue. Several procedures have been described. Basically, these procedures can be split into four main steps that are described in subheadings (1) tissue preparation, (2) the primary antibodies, (3) the visualization of the target, and (4) enhancement of signals with antibody complexes. In addition, a protocol for alkaline phosphatase will be presented in detail in Subheading 5. The terms primary and secondary antibodies refer to the order in which they are applied to the target. The immunocytochemical procedures are not limited to sectioned... 

Administration of Radioisotopes, Tissue Preparation, and Emulsions Resolution and Quantitative Autoradiography Limitations of EM Autoradiographs Protocols Conclusions References... 

There are major differences in tissue processing protocols with variations in the time of immersion in formalin and alcohols. These variables can be controlled and standardized for the individual laboratory but in the case of tissue blocks prepared in other laboratories such variations may significantly affect the staining of tissue antigens. 

Apart from automating the matrix application process, it is critical to evaluate the resulting matrix crystals and coating for analyte extraction, localization, and effect on tissue architecture. Several reports have shown that even for standard analytes mixed with matrices, there is an uneven distribution of the analyte within the MALDI crystals and that sample preparation influences the resulting distribution [18-20], Thus, other active areas of research are focused on optimizing matrix application and sample preparation protocols. 

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 This instrument has been superceded by more recent models, which are now marketed and distributed by MDS Analytical Technologies (http // www.moleculardevices.com).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... 

Follow protocols for tissue preparation appropriate for culturing in collagen. Protocols for rat DRG harvesting and digestion have previously been described (17) see Notc 1). 

The significant advantages of the described protocol rest in the versatility of probe design, the increased tissue permeability and hybridization efficiency because of the small probe size and, finally, the superior tissue preservation as a result of the rapid freezmg/dehydration and vapor-induced fixation. In addition, tissue preparation with the proposed protocol is compatible with immu-nocytochemistry. It is possible, therefore, to obtain serial consecutive paraffin sections from the same CNS and to combine in situ hybridization with immunocytochemistry for direct comparison of the signal from the two techniques in the same cells. For a more comprehensive and detailed review of in situ hybridization m general, the reader is referred to the excellent recent practical guide by Leitch et eil.(l) and also to the seminar series by Harris and Wilkinson (2). 

Figure 4. Schematic of the tissue sample preparation protocol.

Two variations of conventional SIMS have evolved. In one, known as matrix-enhanced SIMS, the MALDI sample preparation protocol is used to coat the sample surface with an organic acid (e.g., 2,5-dihydroxybenzoic acid) or other suitable matrix material to improve ionization efficiency [24,25], This approach has been used to image brain tissue samples from freshwater snails (Lymnaea stagnalis). Another variation employs a coating of a thin layer of gold or other metals to enhance analytical signals [26], This method, known as metal-assisted SIMS, provides images with improved spatial and chemical resolution. 

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