Sample Fixation

In vitro staining is used to colour cells or structures that have been removed from their biological context. Certain stains are often combined to reveal more details and features than a single stain alone can provide. Combined with specific protocols for fixation, sample preparation and investigative methods such as fluorescence microscopy, these standard staining techniques are used as consistent, repeatable diagnostic tools. 

Commercially available preservatives or fixatives such RNA/aterTM (Ambion), and alcohol-based fixatives such as Omnifix are available. Ten percent neutral buffered formalin is the standard widely used fixative for preserving morphology. Samples should be fixed in 10 vol or more of a formalin- or alcohol-based fixative. For the best fixation, samples should not exceed 1-2 mm in thickness. 

Following fixation, samples can either be viewed as whole mounts, using critical-point drying, or by other methods to dry the sample (Hayat, 1989), or... 

Phase-contrast microscopy provides a fast and reliable way to evaluate the onset of sperm chromatin decondensation and the appearance of nuclei assembled in vitro (Fig. 1). After incubation, a IO-/1I aliquot of the incubation mixture should be taken and immediately fixed with an equal volume of 8% (w/v) paraformaldehyde in 154 mM PIPES-NaOH, pH 7.5, for 5 min on ice. After fixation, samples should be diluted with an equal volume of extraction buffer as specified above. Then, 5-ix aliquots of this mixture may then be mounted for viewing between slide and cover slip. To ensure adequate separation between the slide and cover slip, about 2 /xl of melted bee s wax should be deposited onto each corner of the cover slip before mounting. 

Transmission electron microscopy (TEM) is important to monitor cell-free sperm decondensation and nuclear formation. To prepare specimens for TEM, incubation mixture aliquots were fixed for 45 min in 2.5% (v/v) paraformaldehyde, 3.1% (v/v) glutaraldehyde, 0.02% (w/v) picric acid in 30 mAf NaHP04, pH 7.5. After fixation, samples were embedded in 2% (w/v) low-gelling-temperature agarose and postfixed for 15 min in 1% (w/v) OSO4. Samples may be dehydrated either in ethanol and propylene oxide or acetone and embedded for sectioning in Spurr s low-viscosity epoxy resin (Spurr, 1%9). Before examination, sections of about 70-nm thickness should be stained, e.g., with uranyl acetate and Reynolds lead citrate (Re)molds, 1%3). 

In a medical MDCT scanner, histogram methods were used to evaluate emphysematous regions. After lung fixation, samples of various regions of the lung suspected of being either emphysematous or normal were taken and then scanned via micro-CT. The resultant micro-CT data are shown in Fig. 6.17. 

Microwaves have successfully been used for rewarming of blood for medical appHcations (157). Another successful appHcation, not commetciali2ed as of this writing, is the use of microwave heating for rapid tissue fixation (158,159). This procedure appears to reduce the time for tissue sample analysis... 

Fixation tissue samples for immuno histochemistry were fixed in 2% paraformaldehyde, 0.25% glutaraldehyde and 3% sucrose buffered with 0.05M phosphate buffer pH 7. After incubation for 2 hours at 25 °C and 63 hours at 5°C the specimens were washed 3 x 20 min. in phosphate buffer pH 7. Dehydration was carried out using series of ethanol washings 50, 70, 80, 96% followed by 3 x in 99% (V2 hr in each). After additional treatment with 2x2 hrs in petroleum ether (shellsol D70k, Q7712) and 2 x 2 hrs in paraffin with 7% beeswax, the samples were embedded in paraffin. Cross sections of 12.5 /im were made on a Supercut 2050 Reichart Jung pyramitome. 

Sample preparation for AFM analysis is relatively simple. Generally, a desired amount of sample is absorbed onto a smooth and clean substrate surface, for example, a freshly cleaved mica surface. For example, to prepare a food macromolecule sample for AFM imaging in air, the diluted macromolecule solution is disrupted by vortexing. Then, a small aliquot (tens of microliters) of vortexed solution is deposited onto a surface of freshly cleaved mica sheet by pipette. The mica surface is air dried before the AFM scan. A clean surrounding is required to avoid the interference of dust in the air. Molecular combing or fluid fixation may be applied to manipulate the molecule to get more information. 

Some investigators described artifactual DNA sequence alterations after formalin fixation, when testing DNA samples extracted from FFPE tissues. Williams et al.46 reported that up to one mutation artifact per 500 bases was found in FFPE tissue. They also found that the chance of artificial mutations in FFPE tissue sample was inversely correlated with the number of cells used for DNA extraction that is, the fewer cells, the more the artifacts. However, they mentioned that these artifacts can be distinguished from true mutations by confirmational sequencing of independent amplification products, in essence comparing the product of different batches. Quach et al.47 documented that damaged bases can be found in DNA extracted from FFPE tissues, but are still readable after in vitro translesion synthesis by Taq DNA polymerase. They pointed out that appropriate caution should be exercised when analyzing small numbers of templates or cloned PCR products derived from FFPE tissue samples. 

The quantity of RNA extracted from FFPE cell/tissue sections by the heating and nonheating methods, and extracted from fresh cell/tissue embedded in OCT without fixation, was comparable, showing no significant difference for all yields of RNA by Student s f-test, with the exception of one sample, MDA cells fixed in formalin for 24 h (p < 0.05). 

Standardization of IHC/ICC has been a critical issue for more than three decades, especially with the advances in targeted therapy such as the development of trastuzumab (Herceptin) for advanced breast cancer.51 Nevertheless, standardization is a difficult issue because numerous factors may influence the consistency and reliability of immunostaining results, including fixatives, fixation time, AR, antibody clones, detection system, and interpretation (see Part II). In cytopathology, the situation is even worse due to its variable cell sample preparation techniques. Cytopreparation is. .. the foundation of cytomorphology. 52 We believe it is also the foundation of ICC. Therefore, standardization of ICC needs to start with uniform and reliable cytopreparation. 

We found that solutions of hen egg white lysozyme, bovine ribonuclease A (RNase A), or a 1 2 mol ratio of bovine carbonic anhydrase lysozyme formed opaque gels within 2 min when mixed with an equal volume of 20% NBF.25,26 Multi-protein tissue surrogates comprised of 50% w/v lysozyme and up to four additional proteins have also been formed (Fowler et al., unpublished results). After overnight fixation, the surrogates were firm and sliced easily with a razor blade for sampling. To determine the optimal... 

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