For in vitro studies, the product fraction was diluted with PBS and used as such for further dilutions.
Nunc Maxisorb plates were coated with 100 μl of bovine nasal collagen II (5 μg/ml in PBS) overnight at 4°C, and then blocked (0.01 mol/l PBS/0.05% bovine serum albumin; this solution was used for all further dilutions) for 2 hours at 37°C.
Microtiter plates were coated with antibody of IL-17 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4°C, and then blocked (0.01 mol/l phosphate-buffered saline [PBS]/0.05% bovine serum albumin; this solution was used for all further dilutions) for 2 hours at 37°C.
Some whisky purists (Jim Murray, for example) feel that as it has already been diluted, further dilution is unnecessary.
This solution was used for experiments with further dilution.
Because 1 μl of the extracted RNA solution was used directly for RT PCR, without further dilution, to obtain a homogeneous RNA concentration in all the samples, the band pairs in the different lanes of the gel shown in Fig. 1 have different intensities, reflecting the original RNA concentration corresponding to each tumour studied.
Complexes were formed by combining stock solutions of salts and chelator to 1000× the desired final assay concentration in DMSO, incubated at 37°C for 30 min before further dilution in complete medium for cell treatment.
Complexes were formed by combining stock solutions of HNTMB and respective metal salts (FeCl2, FeCl3, CuCl or CuCl2) to 1000× the desired final assay concentration in DMSO, followed by incubation at 37°C for 30 min before further dilution in complete medium for cell treatment.
Copper, Zn, Ca, Mg, K, Na, Mn, Fe and P were also measured in the digested tibias without (for Cu and Mn) or with further dilution (1 10 for Zn, Fe, K and Na, and 1 1000 for Ca, Mg and P) of the samples with UHP water.
