Slide storage

In our hands, slides were best preserved by storage in 100% ethanol at -20? C or -70? C. FISH and conventional GTG banding worked very well on slides older than 4 1/2 years (so far). By comparison, slides kept in dessicator at room temperature became unusable for FISH in about 1 year or less, but could be used for banding.

Fixative composition and trypsin treatment in G-banding

Slides prepared from the same pellet of origin were resuspended in 1:1, 3:1 and 6:1 methanol:acetic acid or in 3:1 ethanol:acetic acid and G-banded. Cell suspension was dropped both from 1m distance and by touching the drops directly to the glass surface. All slides were aged by baking overnight at 65? C and were banded precisely the same way, using the same banding setting. As reported before results show that less spread chromosomes are shorter and that chromosomes significantly elongate during spreading . It is also well known that the degree of metaphase spreading and the amount of cytoplasmic residuum influence banding quality. The cleaner and the better the chromosomes are spread, the sharper the banding pattern.

Fixative composition influences the contrast of the banding pattern. This is illustrated in figs. 5 and 6, in which the banding pattern of cells resuspended in 1:1 methanol:acetic acid is lighter and has a better contrast (white color is whiter) than slides made using cells resuspended in 3:1 fixative (fig 5 comparison) or in 6:1 fixative (fig 6 comparison). Chromosomes prepared using 1:1 fixative are thinner, which helps banding quality. Slides made using cells resuspended in 3:1 fixative are lighter and have better contrast than slides made with a pellet in 6:1 fixative (compare fig 5 and fig.6 ). In other words, the more acid, the lighter the banding. Cells resuspended in 3:1 ethanol:acetic acid fixative yielded a banding pattern that was darker than cells in 3:1 methanol:acetic acid (not shown). This variation in banding contrast with the composition of the fixative could be potentially very useful in banding slides made from cells of various origins.

Time of trypsin treatment is in close relationship with the time for Giemsa staining. The longer the former, the longer the latter. It is also known that the longer the trypsin incubation, the "wider" or puffy the chromosomes become. Although Giemsa staining in these cases needs a longer time, the banding pattern has an increasingly better contrast between light and dark areas (fig. 6). Especially shorter chromosomes, however, tend to lose their smoothness at their edges due to the longer trypsin digestion (fig. 6) Time of trypsin treatment needs also to be correlated to the length of the chromosomes expected to be banded. Very long, prometaphase chromosomes, require long trypsin times (and longer staining times), whereas short chromosomes require decreased exposure to trypsin and to Giemsa.

Another observation drawn from figure 6 is that the overall quality of the bands seems to be slightly better (sharper bands) when slides are dried at room temperature compared with a heated surface. It is likely that higher temperatures alter the morphology of the chromosomes because of protein denaturation.

Banding of chemically aged slides

The possibility of replacing dry heat aging with chemical aging prior to G-banding was explored (Fig. 7a-d). Slides were aged at 94 C in ethanol for 30 seconds (Fig. 7a), 1 minute (Fig. 7b) and 2 minutes (Fig. 7c) and compared with dry heat for 2 minutes (Fig. 7d). Slides were then subjected to trypsin pretreatment and Giemsa staining, similar to the common protocol. Results showed that banding improved as the slides were aged longer in ethanol, but heat-dried metaphases showed superior banding resolutions.