Table 1.

Troubleshooting table.

Problem	Possible reason	Solution
No signal	Quality of metaphases Slides are over 1 year old	If there is a cell pellet stored, try dropping new slides with better humidity conditions and/or longer hypotonic conditions
	Excess cytoplasm surrounding the chromosomes	Try a stronger pepsin treatment; generally, the pepsin stock solution (slide pretreatment solution 2) can be increased in 5-μl increments
	Probe denaturation temperature	Check the actual temperature of the equipment used to denature the probe
	Slide denaturation temperature	Check the actual temperature of the instrument used to heat the slides (water bath or hot plate). Always check the structural integrity of the slide after pretreatment and denaturation (before hybridization); chromosomes should not appear hollow or too shiny
	Detection reagents are approaching expiration	Always check the date on the detection reagents, as some expire within 3 months of purchase
	Detection reagents were improperly diluted	It is important to not overdilute the detection reagents; check the concentrations
	Antibodies were added in the wrong order	It is important to first add the mouse anti-digoxygenin and then detect that with the anti-mouse fluor
	The wrong filter was used during imaging	Always make sure to use the SKY filter cube for taking SKY images.
Weak signal	The chromosomes were too phase-light from the start	When checking the slide, make sure to choose an area of hybridization that contains darkly stained chromosomes, not light gray ones. If no dark areas exist, try to drop new metaphases at a higher humidity
	Forgot to use RNase and/or pepsin	These steps remove excess cytoplasm and RNA, allowing the probe to make full contact with the DNA on the slide (see Box 1)
	Preannealing time was too extensive	Longer preannealing times allow for unique single-copy genomic sequences to form double-stranded DNA with their complementary strands in the tube, rather than on the slide.
	Wash solutions were stored a long period of time	Always make up fresh solutions, especially wash 1. Make sure the pH is in the range of 7–7.4
High background/low signal specificity	Detection was not performed at the correct temperature	The stringency of the washes can be compromised by a lower temperature; therefore, check the actual temperatures of the water baths
	Slides were not denatured long enough	Always check the appearance of chromosomes after denaturation; they should look as good as when you started the procedure. Try increasing time in 5- to 10-s increments
	Insufficient hybridization time	Try a 72-h incubation
	Excess cytoplasm surrounding the chromosomes	See Box 1
	BSA was not properly washed off	Always check the BSA concentration when making wash solution 4. Also note the temperature of the wash solutions after Step 42