By A. Pullman (auth.), J. W. Gorrod, O. Albano, E. Ferrari, S. Papa (eds.)
cytochemical concepts (ICC) which offer an invaluable adjunct to investigations by means of immunoblotting. a selected benefit of a cytochemical method of the investiga tion of mitochondrial problems is that it permits the mosaic distribution of definite of those defects to be detected, while the tissue homogeniza tion considering traditional enzyme assays or immunoblotting precludes this. one more good thing about MEA or ICC is that basically small quantities of tissue are wanted, that's very important due to the fact that a number of the affected sufferers are babies or babies. the most target of this communica tion is to stipulate ways that those suggestions can be utilized within the analysis and additional research of mitochondrial issues. Reference can be made not just to these occasions within which MEA and ICC provide benefits over common enzyme asays and immunoblotting but in addition to contexts during which the opposite applies. four. 2 fabrics and strategies Muscle biopsies for cytochemical research have been snap-frozen utilizing isopentane cooled to - 150°C in liquid nitrogen. Samples have been saved in heat-sealed polythene packets within the vapour part of liquid nitrogen boxes. four. 2. 1 Microphotometric enzyme assays Frozen sections eight Jlm thick have been minimize utilizing a Reichert-J ung Frigocut cryostat microtome built with motor-driven slicing motion to keep up maximal reproducibility of part thickness. Sections have been picked up on microscope slides and air-dried for 15 min at room temperature.
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Extra resources for Molecular Basis of Neurological Disorders and Their Treatment
Here, we have measured the cellular level of Col and ColV mRNAs in the brain of 26-monthold Fisher rats. The effect of the acetyl-L-carnitine pre-treatment was also investigated. 1 Isolation of mitochondria and nucleic acid extraction Fisher rats, 9 and 26 months old, were used. They were kept in an animal room under controlled conditions up to the moment of use. 4 (medium A), with 10-12 up and down strokes in a Dounce glass homogenizer. The homogenate was centrifuged at 1200g for 5 min. The nuclear pellet was washed once at 1200 g for 3 min and the combined supernatants were centrifuged at 18000 g for 10 min to yield a crude mitochondrial pellet.
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5 Immunoblot analysis of trypsin digestion of PVP protein and OSCP in ESMP and USMP. Immunodecoration with antiserum against 25-27 kDa protein fractions of Fa was carried out as described in Houstek et al. (1988). ESMP or USMP was prepared and treated with trypsin (at the concentrations reported in figure ). A1 identifies the PVP protein; A2 OSCP and A3 does not correspond to any Fa protein. Pv P2 and P3 are the products of tryptic digestion of PVP protein. 0.. ,u 8- Fo Fo·try u :! pSinl USMP 6 +trypsin :iE w Z o ~.