, 1990). The procedure

that we applied has been described previously (Dagerlind et al., 1992), and was used with minor modifications as described by Karlstedt et al. (2001). Briefly, the oligoprobe, complementary to the mouse HDC mRNA (5′-CCGTGTCTGACATGTGCTTGAAGATTCTTCACCCCGAAGGACCGAATCAC-3′), was labelled with deoxyadenosine 5′-triphosphate, [α-33P] at its 3′-end by using terminal deoxynucleotide transferase (Promega, Madison, WI, USA), according to the manufacturer’s instructions. Roxadustat molecular weight Non-incorporated nucleotides were removed by purification with Sephadex G-50 QuickSpin cartridges (Roche). The brain sections, eight per mouse, were hybridized with the probe at 56 °C for 24 h. After a series of high-stringency washes that removed a non-hybridized probe, Kodak BioMax films were exposed to the sections and the 14C-standards for 10 days. Quantitative learn more analysis of the autoradiograms was performed with the mcid 6.0 platform (Imaging Research, St Catharines, Canada). Quantitation was performed with 14C-calibration standards (Amersham) in the linear range of the calibration curve, as described previously (Lintunen et al., 1998). The region of interest was defined as the intensity window with the lower threshold determined as three standard deviations from the mean pixel density distribution of the background area,

and the upper threshold as a maximum value of the calibration value. Average values were used as an intensity measure. The specificity of this probe has been established previously (Karlstedt et al., 2001). The assay used here was a combination of methods for the simultaneous determination

of histamine and 1-methylhistamine levels (Miyamoto et al., 2004), HDC activity (Niimi et al., 1997), and HNMT activity (Scott et al., 1991). The animals (36 male 8-week-old C57BL/6J mice and 30 male 8-week-old CBA/J mice) were kept in groups of three (100 lux at the cage bottom) for 2 weeks before the experiment. Mice were oxyclozanide killed at ZT 4, 8, 12 (lights on), 16, 20 and 24 (lights off) by decapitation, and the following brain structures were collected: medulla oblongata, pons, cerebellum, midbrain, hypothalamus, thalamus, hippocampus, striatum, and cortex, all according to the mouse brain stereotaxic atlas (Paxinos & Franklin, 2004). Brain areas were dissected on ice, and samples were snap frozen in liquid nitrogen and stored at −80 °C prior to analysis. Samples were homogenized with a Vibracell sonicator (Sonics, Newtown, CT, USA) on ice in 10 volumes of the homogenization solution, consisting of 115 μm phenylmethanesulfonyl fluoride, 100 μm dithiothreitol and 100 nm 3-methylhistamine (internal standard) in a 0.1 m potassium phosphate buffer (pH 7.0). Sixty microlitres of the homogenate was immediately mixed with HClO4 (final concentration 0.