Tissue samples washing

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. 

In most cases, fixation may be carried out at room temperature. Duration of formalin fixation depends on the nature and the size of the specimen, and may vary from 15 min to 24 h. Longer fixation may be associated with a partial loss of the antigenicity of the component of interest. After formalin fixation, tissue samples are washed in three changes of the buffered saline (PBS) from 15 min to 2 h, but not longer than 24 hours on the whole, since the formaldehyde fixation is partially reversible. After washing in PBS, specimens may be either snap-frozen in liquid nitrogen for subsequent cryosectioning, or dehydrated and embedded in paraffin or synthetic resin. 

Some tissues require a post-protease digestion fixation by treatment with 4% formaldehyde solution for 5 min. This is followed by two washes for 5 min each in PBS. This step may not be necessary for many tissue samples. 

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). 

Single strands of RNA from a cancerous tissue sample are treated with a fluorescent formula and then dropped on the chip. Some of the RNA strands bind with complementary DNA strands on the chip, while others are washed away. Using a computer to observe areas of brightness on the chip s grid, scientists can determine which genes in the tumor are turned on. 

Mix A solubilized, aqueous tissue sample with an equal volume of ice-cold Soln. A, and then add ice-cold Soln. B up to 3.0 ml. The mixture is left for 10 min in an ice bath. After this centrifuge, leave the acidic solution at 0 °C for 10 min with 4000 x g. Wash the pellet three times by resuspension in ice-cold Soln. C and further centrifugation. 

The determination of all four NFs in bovine muscle tissue was used. The tissue sample was homogenized with MeCN, lipids were removed by washing with dichloromethane ethyl acetate, and the concentrated aqueous layer was further purified by washing with hexane. Recoveries were obtained ranging from 60% to 110%, with RSD < 18% and a separation without interference (140). 

A simple assay was used for the separation and MS detection of TMP in tissue. The frozen pulverized tissue sample was homogenized with chloroform acetone, and the extract was evaporated. The residue was dissolved with MeOH acetic acid, and lipids were removed by washing with hexane. An aqueous layer was injected, and TMP was detected and identified using a thermospray LC-MS system. The MS detection was accomplished in the positive-ion mode with SIM of the ion m/z 291 (178). 

Screening of ENRO, CIPRO, DAN, NOR, FLU, OXO, and NALA in pork muscle was based on HPTLC after the SPE procedure (195). Tissue samples were extracted with MeCN-NaOH, the supernatant dried, and the residues dissolved in dipotassium hydrogen phosphate buffer (pH 7.4) hexane was added. The aqueous phase was cleaned up using the SPE technique. After washing the cartridge, analytes were eluted with MeOH-ammonia solution (75 25). After the preparation step, extracts were spotted and eluted on silica gel plates. The plate is first inspected under UV illumination at 312 nm and then sprayed with terbium chloride. The method was validated at the levels of 15 yug/kg for ENRO, CPRO, DAN, and NOR and 5 yug/kg for FLU, 0X0, and NALA. 

For in situ hybridization, a tissue sample is incubated with a labeled nucleic acid probe, excess probe is washed away and the location of hybridized probe is examined. The technique enables the spatial localization of gene expression to be determined as well as the location of individual genes on chromosomes. 

After solubilisation, the sample is added to a sdntillation cocktail suitable for aqueous solutions. Solubilisation of tissues often produces chemiluminescent materials which can distort measurements of radioactivity. To reduce or remove this problem, it is recommended that samples should be kept in the dark before counting. An expensive alternative to scintillation counting for tissue samples, and one which is only justified when handling highly repetitive samples, is combustion of the sample in an appropriate furnace the 14C and 3H labelled material is converted respectively into C02 and H20 which are collected in suitable wash bottles. 32P label is converted into phosphate which remains in the ash following combustion. 

Owing to its convenience and versatility, the method is especially appropriate for pathohistochemical analysis of large numbers of potentially diseased tissue samples. A series of different monoclonal and/or polyclonal antibodies can be used in a sequence of samples. Only the primary antibody has to be altered for a given sample the other steps (successive introduction of biotinylated second antibody, avidin-conju-gated probe, substrate, wash solutions) are identical. 

A 50-g ground tissue sample was homogenized twice with 100 mL of methanol. The homogenates were centrifuged and the two supernatants combined. A 10 mL aliquot of the extract was diluted with 2 mL of water, the methanol evaporated, and an additional 5 mL of water added. The aqueous sample was adjusted to pH 10.5 with 100 iL of 2 M sodium bicarbonate and extracted twice with 10 mL of ethyl acetate. The ethyl acetate was evaporated to dryness, the residue dissolved in 5 mL of acetonitrile/methanol (9 1), and applied to a Bond Elut acid washed silica cartridge (Analytichem International, Harbor City, CA). After washing the cartridge with small amounts of acetonitrile/methanol (9 1), methanol, and dichloromethane, die ractopamine HCl was eluted with 8 mL of dichloromethane/methanol/triethylamine (84 15 1). The eluate was evaporated to dryness in a rotary vacuum evaporator, the residue dissolved in 2 mL of the HPLC mobile phase, and analyzed by HPLC. 

Rats administered radiolabeled maneb via gavage for 7 days at a daily dose of 100 mg/kg had 0.31% of the label in organ and tissue samples, 0.96% of the label retained in the carcass, and 0.18% originating from intestinal washings as measured 1 day after the last dose. When tissue concentrations were reported in relative amounts of the label as maneb, the distribution of the compound in the tissues were as follows thyroid, 865 mg/kg kidney, 51.6 mg/kg fiver, 24.8 mg/kg spleen, 10.7 mg/kg heart, 8.2 mg//kg fat,... 

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