By A. Pullman (auth.), J. W. Gorrod, O. Albano, E. Ferrari, S. Papa (eds.)
cytochemical options (ICC) which offer an invaluable adjunct to investigations through immunoblotting. a specific benefit of a cytochemical method of the investiga tion of mitochondrial issues is that it permits the mosaic distribution of yes of those defects to be detected, while the tissue homogeniza tion all for 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 is very important considering the fact that a few of the affected sufferers are babies or babies. the most target of this communica tion is to stipulate ways that those concepts can be utilized within the analysis and additional research of mitochondrial problems. Reference might be made not just to these occasions during which MEA and ICC supply benefits over typical enzyme asays and immunoblotting but additionally 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 section of liquid nitrogen packing containers. four. 2. 1 Microphotometric enzyme assays Frozen sections eight Jlm thick have been minimize utilizing a Reichert-J ung Frigocut cryostat microtome outfitted 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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22 A TP synthase and energy transfer ,.... •• . •. • %4 • ! :! 4 Titration of the effect of chemical modification of ESMP by amino acid reagents on oligomycin-sensitive H+. ESMP were preincubated with EFA (_e) at the concentrations reported in the figure (left-hand panel) as described in Guerrieri et al. (1987a) before oxygen induced proton translocations. ESMP were preincubated 2 min with diamide (0-0) or Cd 2 + (11-11) at the concentrations reported in the figure. The oligomycin titration (right-hand panel) was carried out on control (0-0) or ESMP treated with 1 mM EFA (e-e); 5 mM diamide (0-0) or in the presence of 5 ~M FCCP (A-A).
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