Fixed tissue

Rous, R. Jones, F.S. (1916). A method for obtaining suspensions of living cells from the fixed tissues and for the plating out of individual cells. J. Exp. Med. 23,549-555. 

Shi S-R, Cote RJ, Chaiwun B, et al. Standardization of immunohistochemistry based on antigen retrieval technique for routine formalin-fixed tissue sections. Appl. Immunohistochem. 1998 6 89-96. 

Prieto DA, Hood BL, Darfler MM, et al. Liquid TissueTM proteomic profiling of formalin-fixed tissues. BioTechniques 2005 38 S32-S35. 

Faulkner SW, Leigh DA. Universal amplification of DNA isolated from small regions of paraffin-embedded, formalin-fixed tissue. BioTechniques 1998 24 47-50. 

Coombs NJ, Gough AC, Primrose JN. Optimisation of DNA and RNA extraction from archival formalin-fixed tissue. Nucleic Acids Res. 1999 27 el2-el7. 

The new fixative approach presents very large logistical issues, including a long transition period when both the new fixative and formalin would be in use in different laboratories across the world. In addition, archival banks for FFPE tissues would diminish in value and become worthless as techniques adapted to the new fixative tissues. On the other hand, data are... 

A common practice is to fix tissues in a small volume of fluid, much to the detriment of quality. If the fluid volume of the fixative is not adequate, water from the specimen will reduce its concentration sufficiently to slow diffusion. A ratio of 20 1 in the volume of fixative to the volume of the specimen will prevent that from happening. 

Glyoxal-based fixatives work faster than formalin. Small biopsies may be ready to process after only an hour while properly grossed larger specimens are ready in about 6h. Structural detail is remarkable in its clarity (Fig. 12.9). Red blood cells are lysed, but that rarely presents a problem. Eosinophilic granules are reduced in prominence (see below). Special stains work well, except for tests for iron (the mildly acidic pH is detrimental) and the silver detection methods for Helicobacter pylori. Most notably, glyoxal-fixed tissues retain strong immunoreactivity for most antigens. The chemistry behind most of this is known. 

AR techniques commonly used for formalin-fixed specimens do not work on glyoxal-fixed tissues and often damage the sections beyond use. Many of the problems involving immunohistochemistry with glyoxal-fixed tissues stem from trying to treat them and formalin-fixed specimens alike. They are different and must be handled accordingly. 

RaitVK, Zhang Q,Fabris D,et al. Conversions of formaldehyde-modified 2 -deoxy-adenosine 5 -monophosphate in conditions modeling formalin-fixed tissue dehydration. I. Histochem. Cytochem. 2006 54 301-310. 

Becker KF, Schott C, Hipp S, et al. Quantitative protein analysis from formalin-fixed tissues implications for translational clinical research and nanoscale molecular diagnosis. /. Pathol. 2007 211 370-378. 

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. 

Jiang X, Jiang X, Feng S, et al. Development of efficient protein extraction methods for shotgun proteome analysis of formalin-fixed tissues. J. Proteome Res. 2007 6 1038-1047. 

Sompuram SR, Vani K, Hafer LJ, et al. Antibodies immunoreactive with formalin-fixed tissue antigens recognize linear protein epitopes. Am. I. Clin. Pathol. 2006 125 82-90. 

Imam SA, Young L, Chaiwun B, et al. Comparison of two microwave based antigen-retrieval solutions in unmasking epitopes in formalin-fixed tissue for immunostaining. Anticancer Res. 1995 15 1153-1158. 

Figure 19.2 A generalized proteomics work flow for the extraction and identification of proteins in FFPE tissue. Formalin-fixed tissues acquired by sectioning, needle dissection, or laser capture are deparaffinized in xylenes and are rehydrated in graded alcohols. The material is resuspended in buffer which generally contains a detergent/ protein denaturant and the sample is heated to complete the extraction process. The protein extract is reduced, alkylated, and digested with trypsin before protein profiling.

This is an important observation given that plasma membrane proteins are often used as markers of disease. This experiment demonstrated that shotgun proteomic analysis could be successfully performed on microdis-sected, formalin-fixed tissues using the antigen retrieval method with a sensitivity equal to that of analysis of the soluble fraction of a fresh-frozen sample. 

The above-mentioned method is effective in identifying the molecules of detected ions. However, because PVDF film is not permeable to light, it is difficult to observe tissue sections. To resolve this problem, we developed a method to fix tissue sections on transparent film, and then performed MS on those sections.6 We used a conductive film because we expected the ionization efficiency would increase when the electric charge accumulation on the sample was reduced. The film used for this purpose was a polyethylene terephthalate (PET) film with a thickness of 75-125 pm, having a 5 15-nm-thick layer of evaporated oxidation indium tin (ITO) upon it (ITO film). This film is used in touch-panel displays because of its high transparency and superior conductivity. We used it to perform MS/MS for tissue sections and succeeded in identifying multiple proteins from mass spectra.6 Therefore, the further development of this method will enable the application of the mass-microscopic method to observe tissue by optical microscope and to perform tandem mass spectrometry (MSn) at the observation part, simultaneously, enabling the identification of molecules included the part. 

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