Embedding Tissue with Paraffin

Human tissue should be fixed in 10% neutral buffered formalin and then dehydrated for embedding in paraffin. Paraffin is nonaqueous embedding medium, so the tissue blocks must have the water removed or be dehydrated. Dehydration is done in organic solvents such as alcohol, acetone, xylene, or toluene. After dehydration, the tissue blocks are embedded with liquid (warm) paraffin. When cooled, the wax embedded block is sectioned on a rotary microtome. Before immunocytochemistry can be performed on the resulting tissue sections, they must be rehydrated by processing with the same organic solvents back to water. Thus, the dehydration and rehydration steps are needed before immunohistochemistry. 

Performing immunohistochemistry on these rehydrated paraffin sections frequently leads to poor results. In contrast, the same antibodies on paraformaldehyde-fixed and cryostat sections will give good results. The issue with formalin-fixed and paraffin-embedded tissue is that the exposure to formalin and dehydration alters the epitopes in the tissue. As a result, formalin-fixed and paraffin-embedded tissues need additional processing methods, known as epitope retrieval or antigen retrieval. Done before immunohistochemistry, epitope retrieval involves heating the sections in buffer with either an acid or base to allow the antibody to recognize the epitope. Also, the exact process of epitope retrieval can be different for individual antibodies. There are numerous papers and books on epitope retrieval and how to apply the method. 

Traditionally, formalin-fixed and paraffin-embedded tissue is used by pathologists for examining human tissue. This method was first adapted for immunocytochemistry with tissue from research animals. Today, for animal research, there is no requirement to use formalin-fixed and paraffin-embedded tissue methods. Table 4.1 shows the steps necessary for formalin/paraffin immunohistochemistry 

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Figure 5.1 Diagrammatic explanation of standardization of IHC via AR and test battery to achieve a maximal retrieval level by an optimal protocol of AR. The intensity of IHC (axis y) is inversely correlated with the time of formalin fixation (axis x) as indicated by a reduced slope. Three arrows indicate a potential maximal retrieval level that may equalize the intensity of IHC to a comparable result for routinely processed, paraffin-embedded tissues with various time of fixation. Reproduced with permission from Shi et alHistotechnol. 1999 22 177-192.

Suurmeijer AJH, Boon ME. Optimizing keratin and vimentin retrieval in formalin-fixed, paraffin-embedded tissue with the use of heat and metal salts. Appl. Immunohistochem. 1993 31 144-146. 

PAb 240, DO-11, and DO-12 antibodies do not precipitate all of the mutant p53 proteins, suggesting that in some mutant molecules the epitopes remain cryptic. Cryptic epitopes can be exposed by denaturation or through mutations. PAb 240 antibody can be used for wild-type and mutant p53 proteins on fresh or paraffin-embedded tissues with the aid of an appropriate antigen retrieval method. Because PAb 240 reacts with a conformational-dependent epitope in the p53 molecule, this antibody has helped define the occurrence of different conformational forms of the p53 protein. 

Epstein AL, Marder RJ, Winter JN, et al. Two new monoclonal antibodies (LN-1, LN-2) reactive in B5 formalin-fixed, paraffin-embedded tissues with follicular center and mantle zone htrman B lymphocytes and derived tumors./ Immunol. 1984 133 1028-1036. 

Gill et al.21 Archival FFPE spinal cord tissue both paraformaldehyde-fixed frozen rat spinal cord tissue and paraffin-embedded same tissue To establish an optimal protocol for detection of low-abundance protein (NeuN) in human spinal cord FFPE tissue sections, testing three AR solutions of pH 6, alkaline, and acidic buffer, with three heating conditions 95,100, and 105°C Heating FFPE tissue sections in an alkaline buffer yields most effective AR-IHC staining results. 

Compound (Miles Laboratories, Elkhart, IN), snap-frozen, and cut into sections for comparison with paraffin-embedded cell sections (3) FFPE Cell Blocks Six cell pellets were fixed in 10% neutral buffered formalin immediately after harvest, at room temperature for 6,12,24h, 3,7, and 30 days, respectively. For further comparison with the cell model system, recently collected sample of human breast cancer tissues were processed by OCT-embedding and snap-freezing the corresponding routine FFPE block that was obtained from the Norris Cancer Hospital and Research Institute at the University of Southern California Keck School of Medicine (USC). This tissue block was processed routinely (formalin-fixed 24h and processed by automatic equipment). 

Hamatani K, Eguchi H, Takahashi K, et al. Improved RT-PCR amplification for molecular analyses with long-term preserved formalin-fixed, paraffin-embedded tissue specimens. J. Histochem. Cytochem. 2006 54 773-780. 

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. 

Part of contents pertaining to the protein-coated beads is reproduced with permission from our article entitled Protein-embedding technique a potential approach to standardization of immunohistochemistry for formalin-fixed, paraffin-embedded tissue sections published in J. Histochem. Cytochem. 2005,53(9) 1167-1170. 

Ramos-Vara J, Beissenherz M. Optimization of immunohistochemical methods using two different antigen retrieval methods on formalin-fixed, paraffin-embedded tissues experience with 63 markers. I. Vet. Diagn. Invest. 2000 12 307-311. 

A protocol for the light microscope radioautography of Lilium longiflorum pollen tubes labeled with [14C]-proline follows. This protocol, which does not require tissue embedding in paraffin or Paraplast, can be modified for paraffin-embedded tissues see Chapter 2). Thus, by employment of the protocol, together with the preceding introductory information in this chapter, one should be able to derive a protocol applicable to the cells or tissue in question. The performance of the protocol requires approval of an institution s Radiation Safety Officer. An inventory of incoming radionuclides, their presence in secondary containers, and their waste must be carefully recorded. The waste must be further broken down into solid waste, liquid waste, and animal carcasses to aid in its proper disposal. 

C. These freeze-dried sections were dry mounted on microscope slides which had been precoated with either Kodak NTB-3 or NTB-10 emulsion. Other techniques which thawed the frozen section, embedded the tissue in paraffin or dipped the section in liquid emulsion were demonstrated to translocate diffusible compounds. Many other similar attempts have been and are currently being made to localize diffusible compounds by autoradiography at the electron microscope level. 

In view of disadvantages associated with the use of cryosections in immunohistochemistry, the general trend is that most immunohistochemical investigations both in diagnostic pathology and in basic research studies are carried out on formaldehyde-fixed paraffin-embedded tissue sections. 

Target Retrieval Solutions from DAKO with pH 6.0 or pH 9.0 are well-suited for use on formalin-fixed, paraffin-embedded tissue sections mounted on glass slides. Compared with 0.01 M Citrate buffer, pH 6, the use of 0.001 M EDTA buffer, pH 9, significantly improves staining results for many antigens, preserves the morphology better and is especially useful in combination with the Dako EnVision visualization systems. 

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). 

The pellet is now ready for processing in an automated tissue processor. If one is not available, the pellet can be embedded in paraffin by hand, gradually replacing the ethanol with xylene and then gradually replacing the xylene with paraffin. 

If epoxy resin-embedded tissue is used, cut 2- jm-thick sections with a glass knife, mount on APES-coated slides, and dry as described in Note 2. Deplasticize the sections by immersing them in sodium eth(meth)oxide for 15 min (8). Wash the sections twice with equal parts of methanol (or IMS) and xylene, twice with methanol, for 3 min each, and rehydrate. Afterward, the same HIER and immuno-histochemical protocols are employed as in paraffin sections. 

Krenacs, L., Tiszlavicz, L., Krenacs, T., and Boumsell, F. (1993) Immunohistochemical detection of CDla antigen in formalin-fixed and paraffin-embedded tissue sections with monoclonal antibody OlO. J. Pathol. 171, 99-104. 

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