Tissue samples transfer

For the MSn measurement of proteins, that is, the measurement of peptides, one has to denature and reduce proteins in the tissue samples, followed by enzyme digestion. Therefore, protein samples should be treated with trypsin after membrane transfer. Trypsin can be attached to the membrane and can be performed in the same steps as in the matrix coating method. 

Enzyme-Immunoassay. Fish tissue samples for testing were cut into uniform 3mm thick slices with parallel razor blades mounted on a handle. Four discs were then punched out from each slice with a stainless steel borer, 3-mm in diameter, and each disc was placed in a well of a 96-well polystyrene microtiter plate (Flow Laboratories, Inc., Hamden, CT). Samples were washed once with 0.2 ml Tris buffer. After the wash solution was aspirated, each sample was fixed in 0.2 ml of 0.3% H O -methanol fixative for 30 min. at room temperature. Samples were then transferred to clean wells and 0.2 ml of a 1 100 dilution of sheep-anti-ciguatoxin-horseradish peroxidase conjugate in Tris buffer was added to each well. The plate was then incubated at room temperature for 1 hr. The sheep-anti-cigua-toxin-horseradish peroxidase was removed by aspiration, and the tissues were immersed for 5 min. in 0.2 ml Tris buffer. Each sample was transferred to clean wells and incubated for 5 min. at room temperature with 0.2 ml of 4-chloro-l-naphthol substrate. The final steps involved removal of the tissue and addition of 0.015 ml of 3 M sodium hydroxide to stop the enzymatic reaction. Absorbance readings at 405 nm of each well were obtained in the Titertek Multi-skan (Flow Laboratories, Inc., Hamden, CT). 

The extraction from non-fatty tissues is performed as follows The tissue sample (20 g.) is finely mixed and acidified with one milliliter of 5N sulfuric acid, then transferred to a conical flask. To this, ten grams of anhydrous sodium sulfate and fifty milliliters of acetone are added, and the mixture is refluxed on a warm water bath for thirty minutes and then filtered. The residual material is further extracted twice with fifty milliliter-portions of acetone. The acetone layers are combined, concentrated by evaporation on a warm water bath to about twenty-five milliliters and then transferred to a separatory funnel. 

As soon as possible (within 1 h), transfer the cryogenic vial containing the tissue sample in RNAtofeAM to a 4 °C refrigerator. Leave the sample at 4 °C from 4 h to overnight, to allow the RNAtofeAM to penetrate the tissue. 

For isolating all DNA contained in a cell, the cell culture or tissue sample is transferred into a buffer which contains a detergent such as SDS or Triton A-100. The detergent disrupts the cellular walls and dissociates any DNA-protein complexes. RNA molecules contained in the cell extract are broken up by treatment with a ribonuclease. Proteins can be digested by a proteolytic enzyme, most commonly proteinase K. The DNA can then be extracted from the mixture by precipitation with ethanol. Only long nucleic acid chains precipitate, single nucleotides and products of the RNA digestion remain in solution. 

As with FMO, this enzyme has some inherent instability and care should be exercised in processing tissue samples of the presence of this enzyme is an issue. Aldehyde oxidase is a true oxidase, in that it transfers electrons to O2 (to form H2O2). The literature contains numerous reports about (milk) xanthine oxidase subsequent work revealed that this is really xanthine dehydrogenase (Rajagopalan, 1997), which is readily converted to an oxidase by proteolysis or modification of sulfides. 

Add digestion was used as an alternative to the enzyme digestion protocol above. The tissue sample was transferred to a glass test tube and an equal volume of 60% nitric acid (Fisher Scientific) was added. The test tube was placed in a beaker of hot (94°C) water for about 1 hour or until the tissue was completely digested. The samples were then centrifuged and the pellet washed with dilute acid and finally resuspended in 0.1% of the aqueous surfactant FL-70. 

Glutaraldehyde solution is used for the first stage, normally at 4% in buffer, and the pieces of tissue are immersed for 4 h. The buffer is designed for correct osmolarity, ionic constitution, and pH of the living tissue. The tissue is then transferred to buffer and undergoes several washes before being postfixed with osmium tetroxide 1.2% for 2h (these times depend on the size of the tissue sample). After several washes in buffer, the next stage is dehydration. Another common variation is to block-stain with uranyl acetate. 

A cryo-cut microtome equipped with Leica Stainless-Steel Re-usable Microtome Knives was used in the following description. Procedural modification may be needed when different equipment was to be used. Frozen animal tissue slices were mounted onto the microtome chunk that held the tissue in place with the OCT compound. The temperature was slowly brought up to —20°C. The frozen tissue samples were then sectioned into lO-jJtm-thick slices. The 10- xm-thick tissue slices were transferred onto a dry porous Si substrate and slowly brought to room temperature. 

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