Tissue-processing

Additionally, tissues from 6 monkeys (3 controls and 3 killed on postischemic day 5) were embedded in paraffin and processed for routine histological analysis. 

For electron microscopical analyses, the fixative was 2.5% glutaraldehyde followed by 1% osmium tetroxide for 1 h at 4°C, and embedding in an epon-araldite mixture (see below). 

DNA damage was evaluated by the terminal deoxynucleotidyltransferase (TdT)-mediated UTP nick end labeling (TUNEL) assay. Degenerating cells were stained by the Fluoro-Jade dye. 

Antibody against Species, isotype Dilution Marker for (references) Vendor 

Primary Proliferation BrdU Mouse IgG 1 100 DNA synthesis (Packard et al. 1973 Miller and Nowakowski 1988) Becton Dickinson, San Jose, CA, USA 

Incubations of microscope slides with small amount of incubation solution must be done in a closed box with a source of water vapor (moist paper towels) to prevent the small volume of solution from drying. While it is possible to buy boxes, most researchers like to make their own with recycled plastic boxes from lab products. Incubation times are generally 24-48 h. Evaporation of the small volumes is a major problem. Agitation, while needed to shorten the incubation time, is not used because of the danger that the incubation solution will run outside the rings. 

Chamber slides are best for cells that require tissue culture grade plastic as a substrate. Lab-Tek chamber slides come with such a slide and they come in a range of well sizes, but a very practical size is the 4-well plate (Fig. 4.7). The microscope slide base is made of Permanox, the same plastic used for plastic petri dish bottoms so that the cells grow like they do on petri dishes or flasks. The nice thing about the chamber slides is that wells on the slide do not need be removed until after immunocytochemistry processing. The wells can be removed just before mounting a 

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Tanji N, Ross MD, Cara A, et al. Effect of tissue processing on the ability to recover nucleic acid from specific renal tissue compartments by laser capture microdissection. Exp. Nephrol. 2001 9 229-234. 

Two different and possibly complementary approaches have been explored. One utilizes a panel of quantifiable internal reference standards (QIRS), which are common proteins present widely in tissues in relatively consistent amounts.11,22 In this instance because the reference proteins are intrinsic to the tissue they are necessarily subjected to identical fixation and processing, and incur no additional handling or cost, other than synchronous performance of a second IHC assay (stain), such that the intensity of reaction for the QIRS and the test analyte can be compared by IA, allowing calculation of the amount of test analyte (protein) present on a formulaic standard curve basis. The other approach seeks to identify external reference materials and to introduce these into each step of tissue preparation for cases where IHC studies are anticipated in this instance the logistical issues of production, distribution, and inclusion of the reference standard into all phases of tissue processing also must be considered, along with attendant costs. 

Burns J, Hambridge M, Taylor CR. Intracellular immunoglobulins. A comparative study of three standard tissue processing methods using horseradish peroxidase and fluorochrome conjugates. J. Clin. Pathol. 1974 27 548-557. 

Srinivasan M, Sedmak D, Jewell S. Effect of fixatives and tissue processing on the content and integrity of nucleic acids. Am. J. Pathol. 2002 161 1961-1971. 

All steps in fixation and tissue processing involve exchange of fluids in the three-dimensional space of the specimen. At the start of fixation, tissue fluid (mostly water) is inside the specimen, while fixative molecules are on the outside. Ignoring for the moment the actual structure of the tissue and the effect a fixative may have upon it, assume that the specimen is like a porous sponge filled with water. To enter this system, a fixative molecule must replace a molecule of water. Diffusion is the driving force when two different liquids meet, there is a gradual equalization in the distribution of their molecules. Ideally, at the end of the process, the concentration of one in the other will be the same both inside and outside the specimen. 

The foundation of fixation in particular as well as the dynamics of tissue processing can be summarized by four points drawn from this relationship. Anyone experienced in histotechnology can attest to the veracity of each, even if they had not thought about it in these terms. 

Figure 12.3 Hydroxymethyl adducts from formaldehyde fixation form highly reactive imines in the presence of ethanol during tissue processing, giving off a molecule of water (upper). While still in alcohol, imines may either form more complex ethoxymethyl adducts or will cross-link to neighboring reactive groups (lower).

Srinivasan et al.16 provide a comprehensive review of how fixation and tissue processing affect DNA and RNA. Formaldehyde attacks the exocyclic nitro-... 

Cross-links from tissue processing Some Few... 

When subjected to the solvents used in tissue processing, immunoreactivity got progressively weaker after each successive exposure to alcohol and xylene, with little change noted after the paraffin. The greatest change in immunoreactivity came at the transition to the completely hydrophobic environment of xylene, probably because it makes it harder to get water back into the protein prior to staining. By the time specimens reached paraffin, heat did little further damage because macromolecules were so drastically modified. 

Grizzle WE, Stockard CR, Billings PE. The effects of tissue processing variables other than fixation on histochemical staining and immunohistochemical detection of antigens. J. Histotechnol. 2001 24 213-219. 

Otah D, Stockard CR, Oelschlager DK, et al. The combined effects of formahn fixation and individual steps in tissue processing on immunorecognition. Biotech. Histochem. 2009 84 223-247. 

Herman GE, Elfont EA, Chhpala EA, et al. Zinc formahn fixative for automated tissue processing. I. Histotechnol. 1988 11 85-89. 

The whole tissue processing from fixation to embedding in paraffin can be performed manually or automated by means of processing machines. Cutting of paraffin-embedded tissues is performed by means of microtomes. Trimmed paraffin blocks are cut at 3 10 pm (5 pm is commonly used). 

Tissue processing tissue specimen (0.5 1.0 mm3) are fixed in 4% buffered formalin for 30 60 min, post-fixed in 1% osmium tetroxide in cacodylate or phosphate buffer, pH 7.2 7.4, stained en bloc for 30 min with 2% aqueous uranyl acetate, then dehydrated in ethanol and embedded in epoxy or acrylic resin. 

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