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Memories Right from MAP2K7-Analysts Which Have Acheived Success

Seven days after the final treatments, animals were terminally anaesthetised (Sagatal; 100?mg/kg i.p.) and were perfused transcardially with 0.1?M PBS. The brains were removed and post-fixed for 7?days at 4��C in MAP2K7 4% paraformaldehyde and then cryoprotected in 30% sucrose containing 0.05% sodium azide and stored at 4��C until the tissue had equilibrated. Coronal sections of tissue (40?��m) throughout the level of substantia nigra were cut using a freezing microtome (Leica 2000R, Nussloch, Germany) and stored as sequential sections in 0.1?M PBS containing 0.05% sodium azide at 4��C and for immunohistochemistry. Every 6th section throughout the SN was selected for TH immunohistochemistry and Nissl staining such that each brain was represented by 12 anterior to posterior sections throughout the level of the SN for TH immunohistochemistry. TH immunohistochemistry was performed as described above. Free-floating sections were mounted onto poly-l-lysine coated slides (Menzel-Glaser, Braunschweig, Germany) and dehydrated for 2?min each in 100%, 98%, and 70% ethanol. They were rinsed in distilled water and stained with cresyl violet (0.5%, Sigma) compound screening assay for 2?min. They were then rinsed in distilled water and further dehydrated in 70%, 98%, and 70% ethanol before being cleared in Histoclear (BDH) for 2?min and mounted with coverslips in dibutyl pthalate/Xylene (DPX) mounting medium (BDH). Tyrosine hydroxylase positive neurones and Nissl-stained cells in the SN were counted stereologically using a Zeiss Axioskop-2 MOT microscope and a DAGE-MTI CCD-100 camera (DAGE-MTI, Michigan City, MI, USA) with StereoInvestigator software (v9.0, MBF Bioscience, Chicago, IL, USA). KU-57788 solubility dmso The SN was manually outlined at 2.5�� magnification. The total numbers of TH positive neurones with normal morphology were counted at 40�� magnification using unbiased optical fractionator estimates for every 6th section throughout the SN. Areas were randomly sampled using a 170?��?170?��M grid size, 100?��?55?��M counting frame size and a dissector height of 14?��M. The total numbers of Nissl-stained cells were estimated at 63�� magnification using unbiased optical fractionator estimates for every 12th section throughout the SN. The settings for Nissl estimates were obtained using a 270?�� 270?��M grid size and a counting frame size of 60?��?30?��M. Coefficients of error were calculated according to (West et?al. 1991) where values less than 0.10 were accepted. Slides were coded in order to blind the investigator to treatment group. Data were analysed using a one-way or two-way anova and post-hoc Dunnett��s test or Newman�CKeul��s test for comparisons were made between controls and treatment groups. In the investigations using stereological analysis data were analysed using a one-way anova and post-hoc Newman�CKeul��s test. All data were expressed as mean?��?SEM and p?<?0.05 was considered to be significant.</div>
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