Fresh Frozen Tissue

We performed a study to apply the a-CGH technique to test the quality of DNA extracted from FFPE tissues by different methods, using a nonheating protocol, a heat-induced extraction protocol, based on AR as applied to IHC, and comparing the findings to extracts from paired fresh frozen tissue samples (unpublished data). The study was conducted in two stages. 

A study of by Palmer-Toy et al.,12 summarized in Table 19.1, provides further empirical evidence of the utility of techniques coupling heating with efficient protein extraction for the proteomic analysis of FFPE tissue. A specimen from a patient with chronic stenosing external otitis was divided in half and preserved as fresh-frozen tissue or FFPE. Ten micromolar sections of the FFPE tissue were vortexed in heptane to deparaffinize the tissue and were then co-extracted with methanol. The methanol layer was evaporated, and the protein residue was resuspended in 2% SDS/lOOmM ammonium bicarbon-ate/20mM dithiothreitol (DTT), pH 8.5 and heated at 70°C for lh. After tryptic digestion, 123 total confident proteins were identified in the FFPE tissue, compared to 94 proteins identified from the fresh-frozen tissue. Hwang et al. also reported up to a fivefold increase in protein extraction efficiency for samples extracted in a Tris-HCl/2% SDS/1% Triton X-100/1% deoxycholate solution at 94°C for 30 min versus samples extracted in 100 mM ammonium bicarbonate/30% acetonitrile at the same temperature.14... 

Figure 20.4 Venn diagram of proteins identified in soluble and insoluble extractions from fresh-frozen tissue and from antigen retrieval from FFPE tissue. Reproduced with permission from Reference 14.

For imaging MS analysis, animals were sacrificed, and the extirpated brains were immediately frozen in powdered dry ice and stored at -80°C until needed. Briefly, fresh-frozen tissues were sliced into 5-pm-thin sections and mounted... 

If a slice of fresh (frozen) tissue is examined directly, little is seen because most of the atoms found in cells are of low atomic mass and scatter electrons weakly and uniformly. Therefore, thin sections must be "stained with atoms of high atomic mass, e.g., by treatment with potassium permanganate or osmium tetroxide. Tissues must also be "fixed" to prevent disruption of cell structures during the process of... 

Sections (7 xm thick) of freshly frozen tissues are mounted on silane-coated slides and fixed with 4% buffered formaldehyde (pH 7.0) for 20 sec (Richter et al., 1999). The sections are rinsed in TBS (pH 7.4) for 15 sec, followed by incubation with EPOS antibody for 3 min at 37°C in an incubation chamber. They are rinsed twice for 15 sec each in TBS, and then developed with peroxidase-DAB detection kit (Dako) in a microwave oven (500 W) for 1 min during microwaving, the slides are cooled by a cold water bath (Werner et al., 1991). After being rinsed in tap water, the sections are counterstained with hematoxylin for lOsec. They are rinsed in tap water and cover-slipped. 

Shapiro C, Crosby K, Lewis M et al. Optimization and Validation of Activation-State Specific Antibodies for the Immunohistochemical Analysis of Fresh Frozen Tissues and Cells. Proc Am Assoc Cancer Res. 2006. 

The following descriptions and protocols are focused on IHC techniques for archival paraffin-embedded tissue sections. The basic principles and protocols for fresh frozen tissue sections are the same as those for paraffin section, except that the AR and dewaxing procedures are not required for frozen tissue sections. Titrations (dilutions) may also differ and must be separately optimized. 

Reverse transcription and PCR amplification can be performed as a two-step process in a single tube or with two separate reactions. RT-PCR performed on fresh-frozen tissue provides high-quality amplification and reliable results. However, when FFPE tissue is used for RT-PCR analysis, the results vary and depend on the level of RNA degradation and length of PCR amplification. To attain a more stable RT-PCR amplification from FFPE tissues, it is typical to choose a target that is less than 150 to 200 nt long. 

Micke P, Ohshima M, Tahmasebpoor S, et al. Biobanking of fresh frozen tissue RNA is stable in nonfixed surgical specimens. 

Fresh frozen tissue - samples with no fixation are frozen and sectioned gives distorted the morphology because no cross linking fixative and components of the tissue can wash out of the section. 

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