Formalin-fixed paraffin-embedded samples

If a monoclonal antibody was generated by immunization with a full-length native protein rather than a peptide, then the immunized mouse will generate antibodies that recognize both linear and conformationally dependent epitopes. Only a small subset of these monoclonal antibodies will likely be useful for clinical use on formalin-fixed, paraffin-embedded tissue (FFPE) samples. Those that are useful tend to have epitopes that are linear the epitopes are not dependent on the protein s three-dimensional conformation (see Chapter 16). Therefore, for antibodies generated in response to immunization with full-length proteins, the peptides that serve as positive controls will be linear stretches of amino acids derived from the native protein sequence, as listed in protein databases. 

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. 

The sample materials from which proteins for proteomics studies may be extracted include fresh or snap-frozen cells from varied sources such as biological fluids, (serum, urine, plasma) and solid tissues such as biopsy specimens. Moreover, proteins isolated from ethanol-fixed paraffin-embedded tissues can be utilized for MS analysis.2 Protocols for the identification of proteins from formalin-fixed paraffin-embedded (FFPE) tissues have been recently developed.3 4 FFPE materials are the most common forms of biopsy archives utilized worldwide, and represent an important advancement for the large-scale interrogation of proteins in archival patient-derived materials. Finally, laser capture microdissected tissues have been successfully used for MS analysis.45... 

Formalin-fixed, paraffin-embedded clinical samples (see Note I). 

Any archived formalin-fixed, paraffin-embedded tissue blocks can be used for preparing tissue sections. However, there are cases that would never demonstrate successful BISH (or FISH) signals. Since, in general, information of how tissue samples were processed for paraffin embedding is very difficult to obtain, the exact cause of unsuccessful BISH assays cannot be clearly identified. The delay of placing tissue samples in a fixative is known to create difficulties of successful molecular histopathology assays. Tissue sections should be cut at 4-5 Xm and placed onto SuperFrost Plus slides (Erie Scientific Company, Portsmouth, NH, USA). The tissue sections can be stored in a slide box at room temperature. 

Scicchitano MS, Dalmas DA, Bertiaux MA et al. Preliminary comparison of quantity, quality, and microarray performance of RNA extracted from formalin-fixed, paraffin-embedded, and unfixed frozen tissue samples. JHistochem Cytochem 2006 54 1229-1237. 

Chromosomes will appear fuzzy and faintly counterstained, nuclei will appear ghost-like, and central areas of DNA may even be removed from nuclei. Under-denaturation will lead to poor signal strength, with strong counterstaining of well-defined chromosomes and nuclei. From this point of view, formalin-fixed, paraffin-embedded tissue is the hardest to work with. Table 4 shows some of the parameters we have used on a series of breast samples. 

Those interested in using immunohistochemistry to study apoptosis need to consider the method of tissue preparation and fixation. Since many antibody epitopes do not survive formalin/glutaraldehyde fixation or paraffin embedding, investigators should determine under what conditions the antibody of interest will work prior to sample collection. There are antibodies that will successfully bind to formalin-fixed, paraffin-embedded material, but if the investigator is unsure, fresh snap-frozen samples can be used to optimize conditions for success since freezing generally will not alter epitopes. 

Immunohistochemistry has also been used to identify Cryptosporidium,Entamoeba histolytica, Trypanosoma cruzi,babesia, Giardia lamblia, Plasmodium falciparum, and P. vivax in fatal cases of malaria in formalin-fixed paraffin-embedded tissue samples. 

Torres-Velez FJ, Nace EK, Won KY, et al. Development of an immunohistochemical assay for the detection of babesiosis in formalin-fixed, paraffin-embedded tissue samples. Am J Clin Pathol. 2003 120 833-838. 

Expression of CD19 is found in the majority of B cell-derived malignancies, as well as on follicular dendritic cells. Antibodies have been developed to detect this antigen in formalin-fixed, paraffin-embedded material. The antibody is quite robust and has excellent correlation with flow cytometric determination of this antigen on both mature and immature B cell processes and performs well in decalcified bone marrow biopsy samples. CD 19 is a potential target for monoclonal antibody therapy, and preliminary data have demonstrated its effectiveness in B-cell depletion, making this an attractive therapy for autoimmune disorders and treatment of malignant B-cell lymphomas. 

Effusion samples Immunocytochemical stains should be performed on formalin-fixed, paraffin embedded cell block sections. 

Commercially available kits are successfully used to recover RNA and DNA from different sample types, including formalin-fixed paraffin-embedded tissue. 

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