Products and methods for tissue preservation
A technology of organization and composition, applied in the direction of chemical method analysis, preparation of test samples, material inspection products, etc., can solve problems such as changes
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Embodiment 1
[0090] sample fixation
[0091] Various samples were immobilized using different compounds and methods disclosed herein. These samples will be used to compare different resistance to polynucleotide degradation characteristics of the present invention.
[0092] Fixation is the first step in any procedure for preserving tissue for histological studies. Utilizes commonly used fixatives to kill the tissue, any bacteria present in the tissue, and to cross-link sample proteins. Commonly used fixatives include: buffered formalin, buffered isotonic saline containing 4% formaldehyde, Bouin's fluid, picric acid, and Carnoy's fixative. These fixatives will be used in conjunction with the reagents described herein to prevent degradation of the polynucleotides in the sample.
[0093] Next is dehydration, or the removal of water from the tissue and replacement with ethanol. A range of different grades of water and ethanol mixtures will be used, typically from 50%-70% to 100% ethanol. T...
Embodiment 2
[0098] Place embedded tissue on a microscope slide:
[0099] Paraffin-embedded tissue from Example 1 was trimmed into a trapezoidal shape and placed in the chuck of a microtome. A microtome is a mechanical device that advances the tissue a fixed amount (1-10 mm) as it moves the block up and down so that the block passes through a blade that cuts the paraffin and tissue into thin sections. When done correctly, successive (series) slices form ribbons.
[0100] The paraffin strips were then transferred to a storage box or directly to microscope slides which had been coated with ovalbumin by means of a small brush. Albumin acts as an adhesive and adheres the section to the slide. Compounds of the invention can be added to the adhesive to prevent degradation of polynucleotides on glass slides.
[0101] The slides are then placed on a warmed tray, and distilled water is added to float the paraffin sections, which are then allowed to swell and cleared. Excess water was removed, s...
Embodiment 3
[0103] Using GenElute from Sigma-Aldrich TM Mammalian Genomic DNA Miniprep Kit (Product No. G1N10) for DNA extraction from formalin-fixed, paraffin-embedded tissues
[0104] Small sections (approximately 20 mg) of paraffin-embedded tissue processed as described herein were placed in 2 ml microcentrifuge tubes. Add 1200 μl xylene. Samples were vortexed for 30 seconds. Centrifuge samples at full speed for 5 min at room temperature. The supernatant was next removed by pipetting without removing any precipitate. Add 1200 μl of ethanol to the pellet to remove residual xylene. Samples were next mixed by vortexing and centrifuged at full speed for 5 min at room temperature. Carefully remove ethanol by pipetting without removing any precipitate. Perform a second ethanol wash. The open microcentrifuge tube was then incubated at 37°C for 10-15 minutes to remove any residual ethanol by evaporation. Digest the tissue by resuspending the tissue pellet in 180 μl of Lysis Solution T....
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