Washing and 12.
detection
Digital image 15.
analysis
286
JosE LUIS FERNANDEZ, VICENTE GOYANES, and JAIME GOSALVEZ
Probe hybridisation 11. All kinds of probes may be hybridised (whole genome, chromosome
painting or subpainting, YACs, satellite, euchromatic, cosmids, etc.) as
usual, overnight, in a moist chamber.
Note: To avoid gel damage the use of rubber solution is not recommended for sealing glass coverslips.
The slides are incubated in cold isotonic saline solution for coverslip
removal. Washes are performed in accordance with the necessary
stringency of the probe, with very gentle agitation.
13. Blocking, fluorochrome conjugated antibody and antibody washes are
performed as set out in current protocols. No amplification of the signal is necessary since protein removal improves probe and reagent accessibility. Directly labelled probes may be used.
14. Cells are DAPI counterstained (lllg/mi in Vectashield, Vector), and
signals visualised with an epifluorescence microscope using 100 x
or 40 x objectives and appropriate fluorescence filters.
Images are captured using a high-sensitivity CCD camera. In our case
we use an Ultrapix 1600, which distinguishes over 16,000 grey levels,
and allows subtraction of the dark current image and corrections for
non-uniform sample illumination. Groups of 50-150 16 bit digital
images are taken in similar conditions, stored in the fIle format of
the camera (.ap£) and then exported to .img fIles.
16. Image analysis is performed using a routine designed with the Visilog
5.1 software. This allows for thresholding the area of interest, background subtraction and measurement of the informative parameters.
These data are exported as .txt files to Excel software for statistical
analysis.
Results
As the dose of ionising radiation increases, DNA breakage level and alkalilabile sites increase, and more ssDNA is generated by the alkaline unwinding solution. Thus, more probe hybridises and fluorescence increases. The
probe selects the chromatin area to be analysed (Figs. 2, 3a-c). An inverse
result would be expected if repair were allowed for. Three simple parameters may be used for quantification of the FISH signal:
detection
Digital image 15.
analysis
286
JosE LUIS FERNANDEZ, VICENTE GOYANES, and JAIME GOSALVEZ
Probe hybridisation 11. All kinds of probes may be hybridised (whole genome, chromosome
painting or subpainting, YACs, satellite, euchromatic, cosmids, etc.) as
usual, overnight, in a moist chamber.
Note: To avoid gel damage the use of rubber solution is not recommended for sealing glass coverslips.
The slides are incubated in cold isotonic saline solution for coverslip
removal. Washes are performed in accordance with the necessary
stringency of the probe, with very gentle agitation.
13. Blocking, fluorochrome conjugated antibody and antibody washes are
performed as set out in current protocols. No amplification of the signal is necessary since protein removal improves probe and reagent accessibility. Directly labelled probes may be used.
14. Cells are DAPI counterstained (lllg/mi in Vectashield, Vector), and
signals visualised with an epifluorescence microscope using 100 x
or 40 x objectives and appropriate fluorescence filters.
Images are captured using a high-sensitivity CCD camera. In our case
we use an Ultrapix 1600, which distinguishes over 16,000 grey levels,
and allows subtraction of the dark current image and corrections for
non-uniform sample illumination. Groups of 50-150 16 bit digital
images are taken in similar conditions, stored in the fIle format of
the camera (.ap£) and then exported to .img fIles.
16. Image analysis is performed using a routine designed with the Visilog
5.1 software. This allows for thresholding the area of interest, background subtraction and measurement of the informative parameters.
These data are exported as .txt files to Excel software for statistical
analysis.
Results
As the dose of ionising radiation increases, DNA breakage level and alkalilabile sites increase, and more ssDNA is generated by the alkaline unwinding solution. Thus, more probe hybridises and fluorescence increases. The
probe selects the chromatin area to be analysed (Figs. 2, 3a-c). An inverse
result would be expected if repair were allowed for. Three simple parameters may be used for quantification of the FISH signal: