Frozen fixed tissue

Frozen fixed tissue is sectioned in a cryostat also known as a microtome in a freezer. The tissue is fixed in 4% paraformaldehyde, rinsed, and then cryoprotected by infiltration in 20% sucrose in buffer with agitation overnight at 4°C (cold room). After 24 h the tissue blocks will sink in the solution, indicating that they are infiltrated. This infiltration step is critical. If skipped, the tissue will freeze with damaged cells and holes from ice crystals, and the resulting tissue sections on microscope slides will be brittle and might crack. The tissue must be fixed first to hold the cells in place, as cryoprotection and freezing without fixation will destroy the tissue. 

ALDEHYDE-FIXED FROZEN CELL/TISSUE SECTIONS... 

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 specificity of antibodies can be exploited in order to probe the in situ organization of cells and tissues. Cellular antigens can be identified both in viable cells and in frozen or fixed tissue sections. Antibodies are used to identify the appropriate antigen in the section and then the position of this primary antibody may itself be detected either directly if it was initially labelled or indirectly using another secondary antibody or molecule to attach to the antibody (Figure 7.8). Samples need to be carefully washed after addition of the primary or labelled antibody in order to prevent any non-specific reactions. Labels that have been successfully linked to antibodies include the following ... 

Commercial kits are available for DNA extraction from the majority of sources, including blood, mouthwash, plasma, serum, frozen tissue, and formalin-fixed tissue. Most kits work very weU if carried out in accordance with the manufacturer s instructions. In addition, they reduce the hkeh-hood of variabihty in the quality and quantity of DNA between batches of samples. Published protocols for the extraction of DNA from paraffin-embedded tissue are also available (37). 

Not only the type (e.g., the cell clone) and the source of availability of an antibody but also its dilution are important in fully utilizing the effectiveness of an antibody as a powerful tool to detect antigens. The optimal antibody concentration for antigen varies, depending on whether the tissue used is aldehyde-fixed or frozen generally higher antibody concentrations are required for sections of aldehyde-fixed tissues (Fig. 4.1). 

Analysis of antigens in fresh, frozen or fixed tissues sub-cellular localization of antigens in tissue culture monolayers observation of bacterial or parasitic specimens ... 

---