Brain sample preparation

Brain et al. [137] reported a tandem mass spectrometry (MS-MS) procedure by which a direct measurement from an n-pentane extract of a surfactant is possible. This procedure is excellent from the standpoint of sensitivity and simplicity of sample preparation but is not commonly applied because of the need of an MS-MS instrument. 

Probably the most effective use of XRF and TXRF continues to be in the analysis of samples of biological origin. For instance, TXRF has been used without a significant amount of sample preparation to determine the metal cofactors in enzyme complexes [86]. The protein content in a number of enzymes has been deduced through a TXRF of the sulfur content of the component methionine and cysteine [87]. It was found that for enzymes with low molecular weights and minor amounts of buffer components that a reliable determination of sulfur was possible. In other works, TXRF was used to determine trace elements in serum and homogenized brain samples [88], selenium and other trace elements in serum and urine [89], lead in whole human blood [90], and the Zn/Cu ratio in serum as a means to aid cancer diagnosis [91]. 

GC/ECD and GC equipped with a microcoulometric detector have been used to determine heptachlor and heptachlor epoxide in a variety of human tissues, including the liver, brain, adrenals, lungs, heart, kidneys, spleen, and pancreas (Curley et al. 1969 Klemmer et al. 1977 Radomski et al. 1968). Details of a sample preparation method were not reported for GC equipped with a microcoulometric detector (Curley et al. 1969). Sample preparation steps for GC/ECD include homogenization, extraction with petroleum ether or hexane, usually followed by a clean-up procedure (Klemmer et al. 1977 Radomski et al. 1968). Recovery, sensitivity, and precision data were not reported (Curley et al. 1969 Klemmer et al. 1977 Radomski et al. 1968). 

SR activity may be determined from cultivated primary dermal fibroblasts, or from lysates of tissue samples (e.g., liver or brain). To prepare lysates from (nonstimulated) fibroblasts, cells from one confluent 78-cm2 plate are suspended in 1 ml of... 

In addition to the silica-based materials mentioned above, modem polymers are widely used for TTA and QTA sample preparation allowing SPE not impaired by undesirable silanol activities. HLB Oasis (Waters) is the tradename for a hydrophilic-lipophilic balance reversed-phase sorbent enabling lipophilic interaction to benzene moieties and hydrophilic interactions to pyrrolidone groups as present in the macroporous copolymer of poly(divinylbenzene-co-iV-vinylpyrrolidone). Elution of analytes is often performed with solvents containing MeOH or ACN. Applying this adsorbent TA such as atropine and scopolamine were extracted from human viscera [15], human serum [97-99], human urine [12] as well as from rat plasma and brain microdialysate [77], Furthermore, this hydrophilic-lipophilic balance phase was also suitable for extraction of the QTA trospium from human and rat plasma [77, 84] and methyl scopolamie from rat plasma [77] (Table 4). 

The sample preparation procedures for the direct analysis of small molecules in tissue have been described by several papers [120-124], Tissues (brain, heart, lung, kidney, liver, etc.), were immediately frozen and stored at -80 °C after harvest. The frozen tissues were subsequently cut into serial 10-20 pm thick section which was typically prepared by cryosectioning on a microtome at a temperature of -20 °C. The adjacent sections were gently mounted onto a conductive surface, MALDI imaging target plate or glass slides. These plates were desiccated under low vacuum for a short period of time until dry, then robotically or manually coated with the... 

The smallest vertebrates are homogenized whole and most laiger animals can be dissected and different tissues removed for storage in 1.5-ml Eppendorf microtubes at — 70° until homogenized. Heart, kidneys, or liver are sufficient to score more than 20 proteins testes, spleen, brain, and muscle can be sampled for tissue-specific enzymes. Additionally, blood, muscle, and/or saliva from most vertebrates can be conveniently sampled without killing the animal. During all sample preparation steps, keep tubes on crushed ice. 

The recovery has been assessed for this assay in the following way 5 replicates of all three QC levels have been prepared according to the sample preparation recipe, which is given in this assay. The resulting data are compared to blank brain extracts (again 5 replicates), which were spiked with the analyte to the expected concentration level after the extraction procedure. In this way, the completeness of the extraction process can be determined. 

Samples prepared from rat brain was homogenized and used directly. Care was taken during the dissection to keep the samples on ice. 

The interest of the consortium in developing and testing new tools for proteome analysis was directed to solutions for particular technical problems concerning sample preparation, the 2D PAGE system, protein quantification, and the development of UniClone sets (nonredundant cDNA expression library) from the adult human brain to be used for creating clinically relevant biochips. These... 

Careful sample selection and preparation are the prerequisites for a successful analysis (Boguski and McIntosh, 2003). Postmortem brain samples for our studies were obtained from the M RC London Brain Bank for Neruodegenerative Diseases, Institute of Psychiatry. The AD patients fulfilled the National Institute of Neurological and Communicative Disorders and Stroke and Alzheimer s Disease and Related Disorders Association (NINCDS/ADRDA) criteria for probable AD (Mirra et al., 1991). A definite diagnosis of AD was performed by historical analysis of the brain samples, which was consistent with the CERAD criteria (Tierney et al., 1988). The brain regions temporal, frontal, occipital, parietal cortex, and cerebellum of patients with AD (72.3 7.6 years old) and controls (72.6 9.6 years old) (Seidl et al., 1997) were used for the studies at the protein level. The... 

Tissue Extraction and Sample Preparation. Brain-subesophageal (BR-SOG) complexes were dissected from adult male and female and quick-frozen... 

Mouledous, L., Hunt, S., Harcourt, R., Harry, J.L., Williams, K.L. and Gutstein, H.B. (2003) Proteomic analysis of immunostained, laser-capture microdissected brain samples. Electrophoresis 24,296-302. Nagy, A. and Delgado-Escueta, A.V. (1984) Rapid preparation of synaptosomes from mammalian brain using nontoxic isoosmotic gradient material (Percoll). J. Neurochem. 43, 1114-1123. 

Preparation of Chelates The procedure we use is adapted from that used by Hui et al ( ) to measure copper in rat brain Samples of feces approximately 0 25 g were ashed in a low temperature asher dissolved in concentrated HC1 and applied to Bio-Rad AG-1 anion exchange columns [Mention of a trademark or proprietary produce does not constitute a guarantee or warranty of the product by the U S Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable ] The various metals were then separated by elution with successively lower concentrations of HCl Recovery of metals from the columns was 97 6+2 7% for Fe, 10(H 2% for Cu, and 93 7+1 8% for Zn Each metal fraction was then refluxed separately with TPP in dimethylformamide to form the metal chelate ( ) ... 

Parkin MC, Wei H, O Callaghan JP. Kennedy RT Sample dependent effects on the neuropeptidome eetected in rat brain tissue preparations by capillary liquid chromatography with tandem mass spectrometry. Anal. Chem. 2005 77 6331-6338. 

Discussion. The method presented provides a fast and reproducible means of determining the ascorbic acid content of various tissues. In addition to a high rate of sample handling (12 samples/h for complex tissues such as liver, brain, and adrenal gland and 15 samples/h for plasma), the method requires a minimum of sample preparation and is practical for routine analysis of biological samples. Furthermore, the method utilizes equipment available to each laboratory with a rudimentary HPLC system. 

Wiseman et al. recently reported the procedures of using DESI for ambient molecular imaging of tissue sections [35], Since DESI did not require sample preparation and a vacuum environment for analysis, the overall procedure was very simple. Examples in their work include the imaging of the distribution of clozapine in the sagittal section of the brain tissue in the rats that received a 250 pg dose of clozapine via an intracerebral ventricular injection. The authors claimed that the procedures should also apply to other types of tissues. 

Sample preparation Tissue. Homogenize brain with 4 volumes MeOH, stir for 15 min, centrifuge at 1300 g for 10 min, acidify supernatant with 300 p.L 1 M HCI, evaporate to dryness under reduced pressure, reconstitute with mobile phase, inject an aliquot. Plasma. Extract 1 mL plasma with 15 mL MeOH, stir for 15 min, centrifuge at 1300 g for 10 min, acidify supernatant with 300 p,L 1M HCI, evaporate to dryness imder reduced pressure, reconstitute with mobile phase, inject an aliquot. 

Sample preparation Tissue. Homogenize 20-200 mg brain tissue with 1 mL 1.5 jtg/mL IS in 1% ammonium acetate -I- 1% sodium azide buffer MeCN 99 1, flush apparatus with 1 mL extraction buffer, add 1 mL acetone, shake for 5 min, centrifuge for 10 min. Add sample to an Extrelut-3 SPE cartridge (Kieselguhr), add 1 mL extraction buffer, wait for 10 min, elute with 15 mL extraction solvent. Evaporate the eluate, take up residue in 50 xL MeOH, add 50 p.L water, inject a 10-25 jiL aliquot. Serum. 100 (xL Serum + 1 mL... 

Sample preparation Blood. Hemolyze 25 p,L whole blood with 50 pL water. 25 p,L Plasma or hemolyzed blood + 100 pL 100 pg/mL ranitidine + 100 xL 5 M NaOH + 5 mL dich-loromethane, mix, shake for 10 min, centrifuge at 1650 g for 10 min. Remove 4 mL of the organic layer emd evaporate it to dryness, reconstitute the residue in 100 p-L mobile phase, inject a 25 pL aliquot. (To measime unbound cimetidine in plasma inject 25 pL ultrafiltrate (Amicon MPS-3 centrifree).) Tissue. Brain tissue + 100 pL 50 pg/mL ranitidine + 1 mL saline, homogenize in an ice bath for 1 min, add 100 pL 1 M NaOH, add 5 mL dichloromethane, extract. Remove 3 mL of the organic layer and evaporate it to dr3mess, reconstitute the residue in 100 pL mobile phase, centrifuge at 10000 g, iiyect a 25 pL aliquot. CSF. Inject an aliquot directly. 

Sample preparation 1 mL Blood or brain + 1 mL 50 mM ammonium acetate buifer, homogenize (Polytron PT-1200C), add 4 mL MeCN, vortex, add 1 mL concentrated brine, allow to stand at -5° for 1 h. Remove the organic phase and centrifuge it at 3000 g, filter, inject an aliquot. 

Sample preparation 2 mL Blood or 250 mg liver (homogenized with 3 parts water) or 500 mg brain (homogenized with 3 parts water) + 2 fig SKF-525-A +1.5 mL pH 9.5 ammonium carbonate/ammonium hydroxide buffer + 10 mL hexane isoprop mol 99 1, rotate at 10 rpm for 10 min, centrifuge at 3500 rpm for 10 min. Remove the organic layer and add it to 2.5 mL 0.25 M sulfuric acid, rotate for 5 min, centrifuge at 1500 rpm for 5 min. Remove the aqueous layer and add concentrated ammonium hydroxide to mtike the pH 9.5, add 1.5 mL chloroform, vortex for 15 s, centrifuge at 1500 rpm for 5 min. Remove the organic layer and add 1 drop of 1% HCl in MeOH, evaporate to dryness at 50° under vacuum, reconstitute with 200 jjiL mobile phase, inject a 100 p,L aliquot. 

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