Paraffin tissue sections section cutting

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). 

A number of proteomic studies on archival material have utilized Liquid Tissue (Expression Pathology, Inc., Gaithersburg, MD), a commercial protein extraction kit for FFPE tissue.4,9,25-28 This kit is also based upon HIAR techniques and shares a similar work flow to the methods already discussed. Thin, typically 5-10pM, sections are cut from paraffin tissue blocks, the paraffin is removed, and the tissue deparaffinized and rehydrated in alcohols and distilled water before microdissection. The cellular material is then suspended in Liquid Tissue buffer and heated at 95°C for 90 min. Trypsin is added, and the material is digested overnight at 37°C prior to reduction with DTT and analysis by LC-MS/MS.26... 

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. 

Experimental samples are mainly derived from tissue culture cells, laboratory animals, or human tissues collected from hospitals after surgical biopsies and autopsies. With human and animal tissue specimens, it is important to arrest metabolic processes within 5-10 min of collection in order to preserve mRNAs from degradation by internal enzymatic reactions (26,27). Most hospitals use 10% buffered formalin as a tissue fixative. Subsequently, each tissue slice is trapped in a paraffin block. Series of 4-5-pm-thick sections are cut and mounted on silanated slides. Formalin-fixed archival tissues have been successfully used in in situ PCR and in situ hybridization protocols (28-32). However, the procedure for RNA protection is not always followed. It is often difficult to alter or control the routine procedures of hospitals for the required protection of mRNAs in surgically removed human tissues. 

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. 

Human brain tissues, for example, are fixed postmortem with 10% formalin for 24-48 hr, dehydrated, and embedded in paraffin. Sections, cut with a rotary microtome, are mounted on coated glass slides. The sections are rinsed three times for 5 min each with 0.1 M sodium phosphate buffer (pH 7.4) and then transferred to 10-15 mM sodium citrate... 

For the HercepTest to be valid, it must be performed exactly according to the manufacturer s directions. The tissue must be fixed in 10% neutral buffered formalin or Bouin s solution. Sections of paraffin-embedded tissues should be cut 3-A xm thick and processed in a standard tissue processor. Epitope retrieval should be carried out in a water bath instead of in a microwave oven. Water is used because it provides more uniform and... 

Sometimes tissues are frozen and then sectioned so that they can be cut directly without the need for embedding. In other instances tissues are embedded in a polymer such as methacrylate instead of paraffin after fixation. There are several variations that are used to make tissue sections to view under the light microscope. In addition to staining with dyes, fluorescent molecules can be specifically attached to macromolecules using antibodies. 

Immunoreactivity diminished or destroyed on pre-cut tissue sections. The intensity of immunostaining may be diminished when pre-cut tissue sections are exposed to air. Use freshly cut sections and reseal paraffin-embedded blocks. 29-33... 

Tissue sections too thick. Cut tissue sections thinner. Formalin-fixed paraffin-embedded tissue sections should be approximately 4-6 pm cryostat section [Pg.141]

Histopathological analysis was performed according to a published procedure by Lipkin. The entire cecum, colon, and rectum of two animals, one each from the control group and the MCM-treated group, were removed and fixed with 10% buffered formalin (12 h), 80% ethanol (12 h), and 95% ethanol (12 h). Representative sections were taken, paraffin embedded, and 4-pm sections cut, mounted into glass slides, and stained with hematoxylin and eosin. Five slides were prepared from each tissue, each slide containing five serial sections. The number of epithelial cells per intestinal crypt over 50 intestinal crypts was counted. The number of crypts containing dysplastic epithelial cells per 50 intestinal crypts was counted. 

A EXPERIMENTAL FIGURE 5-43 Tissues for microscopy are commonly fixed, embedded in a solid medium, and cut into thin sections. A fixed tissue is dehydrated by soaking in a series of aicohoi-water soiutions, ending with an organic soivent compatibie with the embedding medium. To embed the tissue for sectioning, the tissue is piaced in iiquid paraffin for light microscopy or in liquid plastic for electron microscopy after the... 

This new definition of immunocytochemistry derives from advances in antibodylabeling methods in recent years. These advances resulted from specific needs in animal research. Initially, formalin-fixed paraffin sections were used for immuno-histochemistry however, results were inconsistent. In most cases, the antibody did not label anything or it labeled too many cells and was dubbed over fixed. This problem led to the development of the epitope retrieval or antigen retrieval methods, where sections of tissue are treated with heat in buffers before antibody incubations. Unfortunately, epitope retrieval methods can be unique from antibody to antibody and also, for the same antibody, from tissue to tissue. Epitope retrieval is complicated and best avoided. For animal research, a simple method was then developed where tissue was fixed in paraformaldehyde and not formalin or alcohol and subsequently frozen sections were cut on a cryostat. This eliminated the steps of dehydration, embedding in paraffin, rehydration after sectioning, and epitope retrieval before antibody incubation. This was a major breakthrough. 

For immunocytochemistry, fixed tissue must be cut in thin sections to be viewed in the light or fluorescence microscope. There are two common ways of sectioning tissue - the cryostat for fixed frozen tissue and the rotary microtome for paraffin-embedded tissue. In animal research, select the sectioning method based on the experimental design. The method that gives the most reliable results and is the simplest should be selected. For immunocytochemistry, the cryostat is a very efficient and reliable method. The rotary microtome of paraffin-embedded material is more complex and problematic. For immunocytochemistry in animal research, the cryostat method is recommended for reasons discussed in this chapter. 

Cut 3-5 tissue sections (each 10-15 pm thick) from the paraffin block. To avoid contamination, a new blade must be used for each tissue block. 

In preparation for paraflSn infiltration water and fats are removed after fixation from the small blocks of tissue by consecutive extractions with alcohol and fat solvents, such as xylene. The tissue blocks are then placed in several changes of molten paraffin. After the displacement of xylene by paraffin is complete, the tissue blocks in paraffin are removed from the oven and hardened by cooling. Sections can be cut easily by a rotary microtome at a thickness of 5 to 7 microns (0.005 to 0.007mm.). When water-soluble carbohydrates are to be studied, it is important to cut and mount the paraffin sections on slides without exposure to water. In ordinary work, the paraffin ribbons are floated on water and lifted on slides for mounting. Paraffin is removed from the tissue sections prior to microchemical tests by consecutive baths in several changes of xylene and alcohols. 

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