35 PRINS: Hybridization and Primed in Situ Labeling in One Step
471
Counterstaining and mounting
a) Blue counterstaining of DNA is obtained by including OAIlM DAPI in
the antifade solution.
b) Red counterstaining of DNA is obtained by including propidium
iodide in the antifade solution. Mount the slide in 20 III of antifade
solution containing 0.5 Ilg/ml propidium iodide.
Signal quantification with the CGH program QUIPS from VYSIS
This program compares the intensity of two samples, a "test" and a "reference". We assign the telomere signals as "test", whereas the DAPI counterstain is chosen as "reference". The advantage of including the DAPI
counterstain in the quantification is that some of the factors causing variation in in situ detection results would be expected to have a somewhat
similar influence on the DAPI staining. The error possibly induced by such
factors as uneven illumination, variable penetrability of the specimen to
reagents, or locally increased background fluorescence could thus be reduced as they would influence both test and reference values, either increasing or decreasing both together, leaving the ratio less affected. The
disadvantage of the approach is that non-homologous chromosome ends
can not be compared directly, as the DAPI staining is not equal on all
chromosomes.
By this CGH-like approach, signal intensities are transformed into ratio profiles. Since the significance of peak heights in individual profiles
would be difficult to evaluate, we always analyze two types of cells together, using one as a standard against which the other can be compared.
For this comparison, we consider it important that the two types of cells
are spread together on the same slide, and thus are treated identically all
the way from spreading to analysis. In experiments where we compare
different cells within a single sample, this is automatically achieved. In
experiments where we compare cells between samples, we mix male
and female cells, so that the two types of cells to be compared can be easily
identified through the presence or absence of a Y-chromosome. Apart
from these adaptations, the program is used as specified by the supplier
for CGH.
Acknowledgements. This work has been supported by the Danish Cancer Society, by the
Meyer Foundation, and by Boehringer Mannheim (now Roche).
471
Counterstaining and mounting
a) Blue counterstaining of DNA is obtained by including OAIlM DAPI in
the antifade solution.
b) Red counterstaining of DNA is obtained by including propidium
iodide in the antifade solution. Mount the slide in 20 III of antifade
solution containing 0.5 Ilg/ml propidium iodide.
Signal quantification with the CGH program QUIPS from VYSIS
This program compares the intensity of two samples, a "test" and a "reference". We assign the telomere signals as "test", whereas the DAPI counterstain is chosen as "reference". The advantage of including the DAPI
counterstain in the quantification is that some of the factors causing variation in in situ detection results would be expected to have a somewhat
similar influence on the DAPI staining. The error possibly induced by such
factors as uneven illumination, variable penetrability of the specimen to
reagents, or locally increased background fluorescence could thus be reduced as they would influence both test and reference values, either increasing or decreasing both together, leaving the ratio less affected. The
disadvantage of the approach is that non-homologous chromosome ends
can not be compared directly, as the DAPI staining is not equal on all
chromosomes.
By this CGH-like approach, signal intensities are transformed into ratio profiles. Since the significance of peak heights in individual profiles
would be difficult to evaluate, we always analyze two types of cells together, using one as a standard against which the other can be compared.
For this comparison, we consider it important that the two types of cells
are spread together on the same slide, and thus are treated identically all
the way from spreading to analysis. In experiments where we compare
different cells within a single sample, this is automatically achieved. In
experiments where we compare cells between samples, we mix male
and female cells, so that the two types of cells to be compared can be easily
identified through the presence or absence of a Y-chromosome. Apart
from these adaptations, the program is used as specified by the supplier
for CGH.
Acknowledgements. This work has been supported by the Danish Cancer Society, by the
Meyer Foundation, and by Boehringer Mannheim (now Roche).