Frozen section method

Besides the methods noted above, study of the microstmcture of the surface using SEM, and techniques for studying the fracture surface using the frozen section method (the method whereby soft green sheet containing plastic is chiUed at low temperature and is cut to achieve a brittle fracture surface) are frequently used. Evaluation using mercury porosimetry where mercury is injected into the sample is an effective technique for comparing... 

Another method is the denaturation of a tissue section with denaturant on the membrane. In this method, the frozen section is thawed and mounted on the membrane. The transferred membrane is washed with 70% ethanol to remove salt and lipid in the tissue and to fix the protein on the membrane. After that, denaturation is processed with the denaturant. Another method is... 

Stumpf WE, Roth LJ. 1966. High resolution autoradiography with dry mounted, freeze-dried frozen sections. Comparative study of six methods using two diffusible compounds H-estradiol and H-mesobilirubinogen. J Histochem Cytochem 14 274-287. 

Immunocytochemical methods have become an integral part of the clinical laboratory, as well as the research setting (see Chapter 50). Clinically relevant specimens ranging from frozen sections and cell-touch preparations to whole-tissue samples are amenable to analysis (see Chapters 9-13). Panels of antibodies have been developed to aid in the differential diagnosis of tumors (see Chapter 51), and automated instrumentation has been designed to speed the handling of numerous specimens (see Chapter 52). 

If a biotinylated secondary antibody is used for the streptavidin-biotin method the following protocol is suitable for frozen sections ... 

Rapid immunohistochemical study of frozen sections is necessary for intraoperative diagnosis in some cases. Rapid immunostaining is also helpful in confirming or excluding tumor clearance in resection margins or in detecting micrometastases in sentinel lymph nodes in breast cancer patients. Two methods to immunostain frozen sections are the enhanced polymer one-step staining (EPOS) system and the EnVision system both systems are detailed later. 

In situ hybridization overcomes the problem of cellular localization, but it is difficult to relate the expression of a particular mRNA to the expression of the functional protein. Moreover, this method is difficult to carry out. Immunohistochemistry, on the other hand, is a relatively simple technique that overcomes these problems by identifying the precise cellular localization of the functional protein. This technique, using paraffin sections, provides information on the ER status of tumors very simply and rapidly. In addition, this approach is superior to frozen section immunohistochemistry, the dextran-coated charcoal assay (DCC) (see page 276), or the enzyme-linked immunosorbent assay (ELISA) for predicting the response to endocrine therapy. 

The immunohistochemistry of ERs has been exhaustively compared with the DCC assay. Review of the literature indicates -85% agreement between these two methods (Allred, 1999). This is true when immunohistochemistry is restricted to fresh-frozen sections. Immunohistochemistry of frozen sections compared with paraffin sections is a more specific test to detect ER-positive tumors with very low tumor cellularity the DCC assay gives false-negative results for such tumors. A number of publications have also reported good agreement between the DCC assay and immunohistochemistry of paraffin-embedded... 

Chilosi, M., Lestani, M., Pedron, S., Montagna, L., Benedetti, A., Pizzolo, G., and Menestrina, F. 1994. A rapid immunostaining method for frozen sections. Biotech. Histochem. 69 235-239. 

No discussion of fixation would be complete without mentioning a few specialized tissue preparations that have been popular in the past. Frozen sections used to be required for some studies, particularly lymph node studies. Their use was due primarily to antigen destruction caused by formalin fixation, or for example, to the need to study a thicker (10 pm) section to study axons in nerve tissue. The introduction of antigen unmasking methods using heated water has largely reduced the need for frozen sections. 

The primary remaining motive for using frozen sections in routine practice is the need for a quick examination that eliminates the time required for fixation, processing and de-waxing. Frozen tissue sections are also used when direct or indirect immunofluorescence is the detection method, in which case formalin fixation can produce weaker results. Frozen sections should be fixed with acetone (room temperature, five seconds) before storing. They are then re-processed in acetone (4 °C, 10 minutes) and then re-hydrated in buffer for five minutes before immunostaining. 

De Waele et al. (9) recommend immunolabeling isolated cells while in suspension. This necessitates centrifugation after each step until a cytocentrifuge preparation is made prior to contrasting. A preferred method in this laboratory is to prepare cytocentrifuge preparations and to immunolabel them as for frozen sections thereby avoiding several centrifugation steps. 

A satisfactory "sequential" double-staining method using mouse monoclonal antibodies has been in routine use in our laboratory for some time. The method is applicable both to frozen sections and to cytocentrifuge preparations. 

This method is better suited to cultured cells grown on cov-erslips or to frozen section on glass slides. It is not well suited to blocks of tissue. 

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