Nonheating Methods

SDS disrupts noncovalent interactions between subunits of a protein, so if a protein has two subunits, two bands will appear. In the absence of SDS, only one band will appear. This reagent and mercaptoethanol reduce protein subunits that are disulfide bonded. This property of SDS may be responsible for protein denaturation. It should be noted that SDS also permeabilizes cells for antibody access to intracellular epitopes. 

Kidney tissue is fixed with paraformaldehyde-lysine-periodate by vascular perfusion (Brown et al., 1996). Tissue slices are further fixed overnight at4°C with the same fixative and stored in PBS (pH 7.4) containing 0.02% sodium azide. They are placed in 30% sucrose in PBS for at least 1 hr, and then surrounded by a drop of Tissue-Tek embedding medium on a cryostat chuck before freezing by immersion in liquid nitrogen. Cryostat sections about 5 p,m thick are cut at a chamber temperature of -25°C, collected on Fisher Superfrost Plus charged slides, and stored at —20°C until use. 

The sections are brought to room temperature, and a wax pen (PAP pen, Kiyota International) is used to trace a hydrophobic circle around each section. They are rehydrated by immersion in PBS for 5 min most of the PBS is removed from the slide with a tissue paper and the sections are then covered with drops of SDS solution (1% SDS in 

The drops are confined to areas where the sections are encompassed by the wax circles. After the slides have been treated horizontally for 5 min at room temperature, the slide is immersed in PBS in a Coplin jar to remove the SDS. The control slide not exposed to SDS is washed in a separate jar to avoid any contact with SDS. The slides are thoroughly washed three times for 5 min each with PBS, completely removing the SDS otherwise, residual SDS will denature the antibodies subsequently applied to the sections. 

A second nonheating epitope retrieval method involves the use of sodium hydroxide-methanol solution. This solution was used successfully for epitope retrieval in sections of formalin-fixed, acid-decalcified human temporal bone embedded in celloidin (Shi et al 1991). This solution is prepared by adding 50-100 g of NaOH to 500 ml of methanol in a brown bottle and mixing vigorously. The solution can be stored for 1-2 weeks at room temperature it is also available commercially (BioGenex, San Ramon, CA). The clear, saturated solution is diluted 1 3 with methanol before use. A wider application of this solution is awaited. 

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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). 

Another indication of the relevance of the Maillard reaction to human nutrition and metabolism was the finding of urinary loss of bound amino acids after infusion of autoclaved sugar (glucose or finctose)—amino acid or peptide solutions. Nonutilizable amino acids were detected in the blood and urine (S38, S39). After these and other reports, parenteral solutions were sterilized by nonheat methods. 

We performed a study to apply the a-CGH technique to test the quality of DNA extracted from FFPE tissues by different methods, using a nonheating protocol, a heat-induced extraction protocol, based on AR as applied to IHC, and comparing the findings to extracts from paired fresh frozen tissue samples (unpublished data). The study was conducted in two stages. 

For the frozen cell/tissue samples, RNA extraction was carried out by using the TRIzol reagent kit. For the paraffin-embedded cell/tissue, RNA extraction was carried out by two methods heating and nonheating using enzyme digestion for comparison. RT-PCR was performed to compare the results. A... 

Combining both heating and nonheating protocols employed in a sequential order were evaluated, but without any advantage (Fig. 3.4). RT-PCR was performed by standard methods, RNA extracted from fresh MDA cells and human tissue of breast cancer with known tested genes was used as positive control, and pure water was used to replace template (cDNA) as negative control for every experiment of PCR. To assure the accuracy of PCR tests, all reactions were performed in triplicate. 

Prior work (3) has indicated that a combination of 3 analytical techniques—loss of weight on ignition (LOI), Karl Fischer titration (KF), and reaction with LiAlH4—results in quantitative data on the amount of acidic OH groups in the zeolite, even in nonheated wet samples. This method discriminates between acidic and non-acidic OH... 

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