Tissue sectioning methods

If the antigen of interest is abundant and additional sensitivity is not required, then a two-step direct method, rather than a three-step indirect method, can be anployed. With the two-step method, the primary antibody is a biotin-labeled mouse monoclonal antibody, which is followed directly by the ABC incubation step. Because of the abundance of human immunoglobulins in tissue sections, methods for staining immunoglobulin often employ biotin-labeled mouse antihuman immunoglobulin reagents. 

Although the novel AR protocol using citraconic anhydride improved the intensity of IHC on FFPE tissue sections for more than half of the antibodies tested, compared to that achieved by other conventional AR protocols, not all antibodies benefitted, which would argue that the citraconic anhydride method does not serve as a truly universal AR protocol. Indeed, many investigators (Table 1.2) have concluded that different antigens may require different specific AR protocols. In this respect, the test battery is a convenient and cost-effective method for assessing the appropriate AR protocol.2,8 Nevertheless, the present data certainly support inclusion of the citraconic anhydride AR method in such a test battery. With respect to the two heating temperatures for citraconic anhydride, the ultimate choice of method for any laboratory may depend on the equipment available. 

In a study involving decalcified FFPE rat joint tissue sections and a variety of AR methods, Wilson et al.32 reported successful application of 0.2 M boric acid at pH 7.0 as the AR solution combining a low-temperature incubation (60°C for 17 h). The principal advantage of this AR protocol was that it minimized lifting or loss of decalcified hard tissue sections from charged slides. Their basic approach for establishing an optimal AR protocol was a test battery as described above. In a separate series of studies, based upon prior... 

Shi SR, Key ME, Kalra KL. Antigen retrieval in formalin-fixed, paraffin-embedded tissues an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections. J. Histochem. Cytochem. 1991 39 741-748. 

Yano S, Kashima K, Daa T, et al. An antigen retrieval method using an alkaline solution allows immunoelectron microscopic identification of secretory granules in conventional epoxy-embedded tissue sections. /. Histochem. Cytochem. 2003 51 199-204. 

Jiao Y, Sun Z, Lee T, et al. A simple and sensitive antigen retrieval method for free-floating and slide-mounted tissue sections./. Neurosci. Methods 1999 93 149-162. 

Recently, the use of AR has extended into several other areas, yielding interesting information for cytology, fresh cell/tissue sections, and fluorescence IHC (fluorescence in situ hybridization [FISH]), in addition to adaptations of the method for extraction of nucleic acids and proteins from FFPE tissues for use with modern methods of molecular analysis. In this chapter, the emphasis is on expanded applications in diagnostic cytology, fresh frozen cell/... 

Chu W-S, Liang Q, Liu J, et al. A nondestructive molecule extraction method allowing morphological and molecular analyses using a single tissue section. Lab. Invest. 2005 85 1416-1428. 

The heat-induced retrieval protocol for extraction of formaldehyde-modified DNA from FFPE tissue sections provides a simple and effective method of DNA extraction from archival tissue samples.25,45 Based on PCR using three primer pairs ranging from 152-541 bp and a real time KTC-PCR analysis, the heat-induced retrieval protocol yields a better quality and quantity of DNA samples extracted from FFPE tissue sections than conventional methods of extraction.24 In addition, this heating protocol may provide an alternative approach for DNA extraction in some cases such as a recent publication by Ferrari et al. mentioned above.34... 

In conclusion, the data described demonstrate the reproducible quality of DNA samples extracted by using a heat-induced retrieval protocol from FFPE tissue sections, based on careful comparison of a-CGH analysis data. This simple and effective DNA extraction protocol may provide an alternative technique for DNA analysis for CGH as well as for other methods of DNA... 

Following the development of successful heating protocols for DNA extraction from archival FFPE tissue sections as described, it required no great leap of imagination to evaluate similar methods for RNA extraction. Analysis of... 

The heating method of RNA extraction was carried out using the same technique as used for DNA extraction from archival tissue sections documented... 

The quantity of RNA extracted from FFPE cell/tissue sections by the heating and nonheating methods, and extracted from fresh cell/tissue embedded in OCT without fixation, was comparable, showing no significant difference for all yields of RNA by Student s f-test, with the exception of one sample, MDA cells fixed in formalin for 24 h (p < 0.05). 

Is there any other approach or concept that can directly measure protein amount in the tissue section Ten years ago, Roth et al.38 documented a novel method, named the Midwestern assay. This method is based on using two chromogens, soluble and insoluble, for the IHC staining process, to produce sequential production of soluble and insoluble reaction products. The soluble IHC product is used to measure the amount of antigen (protein) by spectrophotometry, while insoluble product indicates the localization of protein in the tissue section. Their experimental results demonstrated that soluble reac-... 

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