Tissue preparation paraffin embedded tissu

Stewart NA, Veenstra TD. Sample preparation for mass spectrometry analysis of formalin-fixed paraffin-embedded tissue proteomic analysis of formalin-fixed tissue. Methods Mol. Biol. 2008 425 131-138. 

The method outlined here uses a modification of the Hedley technique (9,10) to prepare nuclear suspensions from the paraffin-embedded tissue samples. Microtome sections are dewaxed, hydrated, and incubated in pepsin with inter-... 

The preparations most often affixed to silane- (9) or poly-L-lysine-coated slides are Carnoy-fixed suspensions of metaphase cells for chromosome studies (see Chapter 47) cytospins of cultured cells followed by methanol fixation sections of formalin-fixed, paraffin-embedded tissues and frozen sections fixed with acetone or ethanol/acetic acid after cutting. Each of these approaches imparts different intracellular effects that may modify cytological detail and/or hybridization. (See Chapters 8 and 9 for a discussion of fixatives and frozen-section preparations.)... 

Preparation of Paraffin-Embedded Tissue Cuiture Ceii Sections... 

Any archived formalin-fixed, paraffin-embedded tissue blocks can be used for preparing tissue sections. However, there are cases that would never demonstrate successful BISH (or FISH) signals. Since, in general, information of how tissue samples were processed for paraffin embedding is very difficult to obtain, the exact cause of unsuccessful BISH assays cannot be clearly identified. The delay of placing tissue samples in a fixative is known to create difficulties of successful molecular histopathology assays. Tissue sections should be cut at 4-5 Xm and placed onto SuperFrost Plus slides (Erie Scientific Company, Portsmouth, NH, USA). The tissue sections can be stored in a slide box at room temperature. 

Proteins can be detected in tissue sections or cell cultures using similar immune detection systems. Use of an antibody to detect specific proteins in tissues is called immunohistochemistry, whereas detection of proteins in cell suspensions is called immunocyto-chemistry. Tissues can be prepared by fixation and embedding in paraffin wax, or by rapid freezing in a compound that inhibits ice formation in the tissue, so as to preserve cell morphology. Some antibodies do not work well with paraffin-embedded tissues, probably because the antibody cannot access the antigen properly (133). The most common labeling system used for detection of the bound antibody is an enzyme-coupled secondary antibody that produces a color reaction... 

To prepare tissue, 2-3 mm slices are removed as quickly as possible into ice-cold acetone and left on ice for 4-6 h. (Less than 4 h can result in incomplete penetration of fixative and subsequent formation of ice crystals.) Samples are then stored m a -20°C freezer until they can be processed using routine paraffin embedding techniques. 

The following procedure is more suitable for routine application than other methods as many as 200 specimens can be processed at a time with this procedure. Sections (2 pm thick) of formalin-fixed and paraffin-embedded tissues are mounted on silane-coated slides. They are deparaffmized with xylene and rehydrated in a series of descending concentrations of ethanol. The sections are immersed in 0.01 M sodium citrate buffer (pH 6.0) in plastic Coplin jars and heated in an autoclave at 120°C for 20min. After the sections have cooled down to room temperature for 20 min, they are incubated in the freshly prepared following silver staining solution for 25 min at room temperature. 

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. 

Histopathological analysis was performed according to a published procedure by Lipkin. The entire cecum, colon, and rectum of two animals, one each from the control group and the MCM-treated group, were removed and fixed with 10% buffered formalin (12 h), 80% ethanol (12 h), and 95% ethanol (12 h). Representative sections were taken, paraffin embedded, and 4-pm sections cut, mounted into glass slides, and stained with hematoxylin and eosin. Five slides were prepared from each tissue, each slide containing five serial sections. The number of epithelial cells per intestinal crypt over 50 intestinal crypts was counted. The number of crypts containing dysplastic epithelial cells per 50 intestinal crypts was counted. 

The method outlined here uses a modification of the Hedley technique (9,10) to prepare nuclear suspensions from the paraffin-embedded tissue samples. Microtome sections are dewaxed, hydrated, and incubated in pepsin with intermittent vortexing and mechanical disruption to release the nuclei. After completion of the tissue digestion, the nuclei are either suspended in 70% ethanol for storage or stained with propidium iodide (PI) for FCM analysis. There are three alternative techniques for preparation of the nuclei on microslides, in tea bags, or in test tubes. 

Samples for identity testing can be any specimen that contains DNA. Samples obtained from an individual for paternity testing or as a reference sample to be compared with DNA prepared from evidence are usually peripheral blood or buccal mucosa. Samples useful for forensic testing, engraftment assays, and the identification of clinical samples may range from plucked hairs to bone marrow aspirates to paraffin embedded tissue. While subject to degradation over time in the presence of enzymes, acidic or basic conditions, or high temperature, DNA is a remarkably stable molecule that can be recovered and successfully analyzed from solutions, surfaces, and cells. 

From large pieces of tissue, paraffin sections of fixed tissue or cryostat sections of fresh or fixed tissues are prepared (Table 11.3). Many laboratories prefer either one or the other. Cryosectioning is rapid and relatively easy but morphology may be better preserved with paraffin sectioning. Methacrylate sections are used rarely for ISH. The sensitivity of ISH on paraffin-embedded tissue sections or on cryosections has rarely been compared (Lum, 1986 Tournier et al., 1987) but does not seem to differ greatly. This may depend on the type of tissue and the nature of the target nucleic acid. 

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. 

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