Formalin fixed and paraffin embedded

Sato Y, Sugie R, Tsuchiya B, et al. Comparison of the DNA extraction methods for polymerase chain reaction amplification from formalin-fixed and paraffin-embedded tissues. Diagn. Mol. Pathol. 2001 10 265-271. 

Castiglione F, Degl Innocenti DR, Taddei A, et al. Real-time PCR analysis of RNA extracted from formalin-fixed and paraffin-embeded tissues effects of the fixation on outcome reliability. Appl. Immunohistochem. Mol. Morphol. 2007 15 338-342. 

Guo T, Wang W, Rudnick PA, et al. Proteome analysis of microdissected formalin-fixed and paraffin-embedded tissue specimens. J. Histochem. Cytochem. 2007 55 763-772. 

Xu H, Yang L, Wang W, et al. Antigen retrieval for proteomic characterization of formalin-fixed and paraffin-embedded tissues. J. Proteome Res. 2008 7 1098-1108. 

Kroll J, Becker KF, Kuphal S, et al. Isolation of high quality protein samples from punches of formalin fixed and paraffin embedded tissue blocks. Histol. Histopathol. 2008 23 391-395. 

APPLICATION OF SHOTGUN PROTEOMICS TO FORMALIN-FIXED AND PARAFFIN-EMBEDDED TISSUES... 

Krenacs, L., Tiszlavicz, L., Krenacs, T., and Boumsell, F. (1993) Immunohistochemical detection of CDla antigen in formalin-fixed and paraffin-embedded tissue sections with monoclonal antibody OlO. J. Pathol. 171, 99-104. 

Berg, D. Molecular profiling of signaling pathways in formalin-fixed and paraffin-embedded cancer tissues... 

Berg D, Wolff C, Malinowsky K et al (2012) Profiling signalling pathways in formalin-fixed and paraffin-embedded breast cancer tissues reveals cross-talk between EGFR, HER2, HERS and uPAR. J CeU Physiol 227 204-212... 

Berg D, Malinowsky K, Reischauer B et al (2011) Use of formalin-fixed and paraffin-embedded tissues for diagnosis and therapy in routine clinical settings. Methods Mol Biol 785 109-122... 

Sections (5 xm thick) of formalin-fixed and paraffin-embedded tissues are placed on slides, dried overnight at 37°C, deparaffinized with xylene, and rehydrated with descending concentrations of ethanol. They are treated with 3% hydrogen peroxide in methanol for 5-10 min and then rinsed with PBS. The slides are immersed in 10 mM citrate buffer (pH 6.0), and heated for 2 min at 100% power, followed by 8 min at 80% power. After being rinsed in PBS, the sections are incubated in an appropriate primary antibody. They are rinsed in PBS and then treated for 4-8 min with egg white avidin solution (2.5 g of egg white avidin in 0.1 mM PBS) (Ventana Medical Systems). Avidin binds to endogenous biotin. 

Sections (4 xm thick) of formalin-fixed and paraffin-embedded tissues are mounted on gelatin-coated slides, which are placed in a beaker containing 1,000 ml of 0.2 M sodium... 

Interobserver variation in the grading of VIN has been observed when using hematoxylin-eosin only, MIB-1 monoclonal antibody alone, or combined hematoxylin-eosin and MIB-1 antibody (van Beurden et al., 1999). This antibody is the most versatile proliferation worker for the sections of formalin-fixed and paraffin-embedded tissues. Normal vulvar skin and VIN lesions are fixed with formalin and embedded in paraffin according to standard procedures. 

The enhanced peroxidase one-step (EPOS) method is considered superior to standard ABC technique in that the former is more sensitive than the latter. It is known that the Ki-67 antibody can only be used on fresh or frozen tissues, whereas the monoclonal antibody MIB-1, developed against a part of the Ki-67 antigen molecule, can be used on sections of formalin-fixed and paraffin-embedded tissues using antigen retrieval. Recently, EPOS Ki-67 antibodies were developed which consist of antibody molecules and horseradish peroxidase bound covalently to dextran (Bisgaad et al., 1993). This method has been applied for localizing PCNA and Ki-67 antigens (Tsutsumi et al., 1995). 

Simultaneous, double immunostaining of two antigens in single cells in sections of formalin-fixed and paraffin-embedded archival tissues can be carried out. This is accomplished by using microwave heating to detect otherwise undetectable nuclear antigens, followed by the labeled avidin-biotin (LSAB) procedure and the alkaline phosphatase (APAAP) protocol to detect cytoplasmic or membranous antigens (Bohle et al 1997). 

Sections (4 xm thick) of formalin-fixed and paraffin-embedded tissues are deposited on a coated slide, deparaffinized, rehydrated, and rinsed in PBS. They are placed in sodium citrate buffer-containing plastic jars and heated twice for 5 min each in a microwave oven. Following cooling at room temperature for 20 min, the sections are treated with 0.3% trypsin. They are washed in PBS, blocked with normal serum for 10 min, and then overlaid with fluorescein-conjugated rabbit antibodies to human IgG at 1 20 dilution in PBS for 45 min at room temperature. 

Double immunofluorescence labeling in conjunction with microwave heating can be used to visualize two markers at the same cellular location in routine formalin-fixed and paraffin-embedded tissue sections (Mason et al 2000). The primary antibodies are either monoclonal antibodies of differing isotype/subclass or antibodies from different species. Labeling is visualized on a conventional fluorescence microscope equipped with a cooled analog monochrome CCD camera (Model C 5985, Hamamatsu Photonics, Billerica, MA) and recorded using off the shelf personal computer hardware and software. Contrary to general belief, paraffin-embedded tissue sections do not show excessive nonspecific fluorescence. 

Sections of formalin-fixed and paraffin-embedded tissues are placed onto silane-coated slides, deparaffinized, and rehydrated. They are placed in 0.1 M EDTA (pH 8.0) and exposed to steam heat for 30 min. The slides are cooled for 5 min, rinsed in tap water, loaded onto the ES Autostainer, and treated with Protease 2 (Ventana) for 8 min. Incubation is carried out in the primary 34 3E12 antikeratin (diluted 1 10 in PBS) for 32 min and in biotinylated secondary antibodies followed by streptavidin for 8 min each. The staining is visualized on the instrument using 3-amino-9-ethylcarbazole. Between each step, the slides are rinsed in Tris-buffered saline. The results of this protocol are shown in Figure 8.8. 

Sections (4 xm thick) of formalin-fixed and paraffin-embedded ovarian tissue are mounted on silane-coated slides and air-dried for 24hr at 3°C (Davidson et al., 2000). They are deparaffinized, rehydrated, placed in 0.01 M sodium citrate buffer (pH 6.0), and heated twice for 5 min each in a microwave oven. 2H5 antibody (PharMingen, Becton Dickinson, San Jose, CA) is used at a concentration of 2 p,g/ml to detect sialyl Lex antigen. Staining is performed with labeled avidin-biotin. Negative controls consist of the exclusion of the primary antibody, while positive controls consist of carcinomas that have been shown to be immunoreactive for the antigen in earlier studies. 

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. 

Presently, nonradioactive probes, especially biotin or digoxigenin, are favored because they are less hazardous to work with, can be more rapidly developed, and provide better spatial resolution. Thus, introduction of nonradioactive detection systems has made ISH, using formalin-fixed and paraffin-embedded tissues, more accessible for application to molecular cell biology and diagnostic pathology. However, radioactive detection systems are more sensitive than nonradioactive probes, especially oligonucleotide probes used instead of cRNA probes (Sperry et al., 1996). 

Ki-67 antigen immunohistochemical staining is a simple and reliable procedure for studying tumor proliferative activity in frozen or formalin-fixed, paraffin-embedded tissues, including archival specimens. This antigen can be retrieved on sections of formalin-fixed and paraffin-embedded tissues by autoclave treatment (Fig. 10.1) or microwave heating (Fig. 10.2). Both methods are reliable and are presented later. 

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