Fig 4 (FISH guide)
LegendFig. 4.Methanol, ethanol and fixative comparisons.Chemical aging, dry heat (3 minutes at 94 C each) and pretreatment were used in various combinations to identify the best sequence of steps allowing both efficient hybridization and DAPI banding. The first column(a, d, g, j) shows slides aged in ethanol, the second column(b, e, h, k) shows slides aged in methanol and the third column(c, f, i, l) slides aged in 3:1 fixative. In the first row(a-c), slides were subjected first to dry heat, then chemical aging and then enzyme pretreatment. In the second row(d-f), chemical aging was followed by dry heat and enzyme pretreatment. In the third row(g-i), dry heat was followed by pretreatment and chemical aging. In the forth row(j-l), chemical aging was followed by pretreatment and dry heat. In all images, the last steps were denaturing and DAPI staining. A comparison of all images showed that chromosomes aged in ethanol preserved the best shape and banding pattern after denaturing (first column). Although dry heat aging is beneficial for banding if performed before protease pretreatment, it is detrimental for hybridization and is not helpful for slides subjected to the denaturing process. DAPI banding is preserved better on chemically aged slides than on heat pretreated slides after exposure to protease pretreatment (compare last two rows).
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Last modified on: Feb12, 2001