Tissue sectioning

Keywords Immunohistochemistry Antibody labeling Fluorescence microscopy Fluorescent immunocytochemistry Fluorescent immunohistochemistry Indirect immunocytochemistry Immunostaining 

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. 

Immunocytochemistry, DOI 10.1007/978-l-4419-1304-3 4, Springer Science+Business Media, LLC 2010 

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Fluorescence Microscope. A useful light microscope utilizes UV light to induce fluorescence in microscopic samples (40). Because fluorescence is often the result of trace components in a given sample rather than intrinsic fluorescence of the principal component, it is useful in the crime laboratory for the comparison of particles and fibers from suspect and crime scene. Particles of the same substance from different sources almost certainly show a different group of trace elements. It is also very useful in biology where fluorescent compounds can be absorbed on (and therefore locate and identify) components of a tissue section. 

Table 11-5. X-ray Emission Spectrography of Biological Tissue Sections. Tabular Summary of Illustrative Information...

Unknown, denoted by superscript U, tissue section of dry prostate. Standard, denoted by superscript St, organic material containing zinc salt in known amount. 

Mineral elements in biological tissue sections, derivation and use of equation for determination, 301-305 Minerals, analysis, use of borax flux by Claisse in, 173, 207, 209 use of curved-crystal spectrograph for small samples in, 206, 207 assay by x-ray emission spectrography, 199-209... 

Tin plate, thickness of tin coating on, determination by x-ray spectrography, 148, 149, 157, 158 Tissues, determination of dry weight by absorptiometry, 297-300 Tissue sections, biological, determination of mineral elements in, 301-305 Titanium, as internal standard in vanadium determination, 188 determination by x-ray emission spectrography, 222, 329 trace analysis by x-ray emission spectrography, 163, 225-229 Topaz, as analyzing crystal, 116-118, 220, 318-327 Total reflection, 112, 117... 

Glycogenosis type VIII (phosphorylase b kinase deficiency) gives rise to myopathy and liver disease, either singly or in combination. Phosphorylase b kinase (PBK) converts the inactive b form of both muscle and liver phosphorylases to the active a forms of the enzymes. The ischemic lactate test sometimes shows a flat result as in McArdle s disease, but is more likely to be normal. Histochemical demonstration of myophosphorylase activity in tissue sections shows a near-normal reaction due to the presence of phosphorylase a. Accumulation of glycogen is modest and found mainly in type 2 (fast-twitch glycolytic) muscle fibers. 

Target tissue sections from animals sacrificed at 8 hr and later after dosing showed the presence of microspheres in the extravas-cular interstitial tissue. Changes in red blood cells and damage to other cellular components suggest that the cytotoxic properties of adriamycin have been retained. The microspheres appeared to still be intact for up to 72 hr. 

Enzyme electrodes belong to the family of biosensors. These also include systems with tissue sections or immobilized microorganism suspensions playing an analogous role as immobilized enzyme layers in enzyme electrodes. While the stability of enzyme electrode systems is the most difficult problem connected with their practical application, this is still more true with the bacteria and tissue electrodes. 

Figure 5.1 Visualization of the distribution of the DA transporter, D3 receptor, and K2-opioid receptor in the human brain of a drug-free control subject and a representative cocaine overdose victim. (A, B) The DA transporter was measured using [3H]WIN 35,428 (2 nM) as described previously. (C, D) The D3 receptor was measured using [3H]-(+)-7-OH-DPAT (1 nM) in the presence of GTP (300 m/W) to enhance the selective labeling of the D3 receptor subtype over the D2 receptor subtype as described previously. (E, F) The K2-opioid receptor subtype was measured using [125l]IOXY on tissue sections pretreated with BIT and FIT to occlude binding to the p- and 8-opioid receptors, respectively.

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. 

Notes CA98°C, heating tissue sections in 0.05 % citraconic anhydride at 98° C for 45 min CAPC, heating tissue sections in 0.05% citraconic anhydride in a plastic pressure cooker using microwave oven for 30 min Low pH, heating tissue sections in acetic buffer of pH 1-2 for shorter time as described in the text Citrate, conventional citrate acid buffer 0.01 M at pH 6.0 with same heating condition of a plastic pressure cooker described above. 

Although the novel AR protocol using citraconic anhydride improved the intensity of IHC on FFPE tissue sections for more than half of the antibodies tested, compared to that achieved by other conventional AR protocols, not all antibodies benefitted, which would argue that the citraconic anhydride method does not serve as a truly universal AR protocol. Indeed, many investigators (Table 1.2) have concluded that different antigens may require different specific AR protocols. In this respect, the test battery is a convenient and cost-effective method for assessing the appropriate AR protocol.2,8 Nevertheless, the present data certainly support inclusion of the citraconic anhydride AR method in such a test battery. With respect to the two heating temperatures for citraconic anhydride, the ultimate choice of method for any laboratory may depend on the equipment available. 

In a study involving decalcified FFPE rat joint tissue sections and a variety of AR methods, Wilson et al.32 reported successful application of 0.2 M boric acid at pH 7.0 as the AR solution combining a low-temperature incubation (60°C for 17 h). The principal advantage of this AR protocol was that it minimized lifting or loss of decalcified hard tissue sections from charged slides. Their basic approach for establishing an optimal AR protocol was a test battery as described above. In a separate series of studies, based upon prior... 

Shi SR, Key ME, Kalra KL. Antigen retrieval in formalin-fixed, paraffin-embedded tissues an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections. J. Histochem. Cytochem. 1991 39 741-748. 

Shi S-R, Cote RJ, Chaiwun B, et al. Standardization of immunohistochemistry based on antigen retrieval technique for routine formalin-fixed tissue sections. Appl. Immunohistochem. 1998 6 89-96. 

Yano S, Kashima K, Daa T, et al. An antigen retrieval method using an alkaline solution allows immunoelectron microscopic identification of secretory granules in conventional epoxy-embedded tissue sections. /. Histochem. Cytochem. 2003 51 199-204. 

Jiao Y, Sun Z, Lee T, et al. A simple and sensitive antigen retrieval method for free-floating and slide-mounted tissue sections./. Neurosci. Methods 1999 93 149-162. 

Recently, the use of AR has extended into several other areas, yielding interesting information for cytology, fresh cell/tissue sections, and fluorescence IHC (fluorescence in situ hybridization [FISH]), in addition to adaptations of the method for extraction of nucleic acids and proteins from FFPE tissues for use with modern methods of molecular analysis. In this chapter, the emphasis is on expanded applications in diagnostic cytology, fresh frozen cell/... 

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