I am currently trying to do FISH on human skin biopsies that have been routinely fixed in formalin and embedded in paraffin. I am using the CEN X/Yq12 probes from ZytoVision and their ZytoLight Tissue Implementation Kit. De-paraffinization was in xylol, isopropanol, 96% and 70% ethanol. The manufacturer's protocol performs a heat pretreatment for 15 min at 98°C in a citrate buffer and continues with a pepsin treatment at 37°C which should be optimized for the tissue and probes. Denaturation was at 75°C in the dark. Hybridization was at 37°C overnight. All (wash) buffers were used from the kit at correct indicated temperatures.
I saw a positive signal for the X probe with 6 minutes of pepsin in a female tonsil test sample. 6 minutes seemed to have also worked for the Y probe in one male skin sample, but no signal for either the X probe or a second skin sample from the same run. Since then, I have tried 2, 4, 6 and 8 minutes of pepsin in more skin samples but haven't been able to get any signal. A second tonsil sample (male) also failed to give a signal with 6 min.
Nuclear counterstain was with ProLong Diamond or VectaShield. Additional CD3 staining worked fine in all cases. X and Y probes are both in the same tube (dual color probe), so it's not possible that I forgot to pipet one. Errors in microscope/laser/filter settings are rather unlikely. Thickness is 2 µm as recommended.
I am desperate for any advice on what could be issue here. I can still try to reduce the heat pretreatment or play around with the pepsin incubation, but I'm running out of time. Any theory or idea for troubleshooting will be highly appreciated!
The most likely issue is that your nucleic acid is crosslinked to much - this is normally due to excessive length in formalin. You may want a more aggressive antigen retrieval, you could heat for longer if needed - we heat for 10 mins @ 121C - as long as morphology doesn't suffer you could be okay. You could also try using a high pH buffer like Tris instead of sodium citrate. Another approach would be to use a microwave or formic acid to uncross link your samples.
Fixation time may vary for sample to sample, but your target probe will also vary by how much it is affected by fixation, its annoying but you need to try and find a good balance.
In some instances, I have used Proteinase K instead of pepsin for the pre-treatment. Also, I have had to use up to 30 min of the heat treatment at times.
It might be worth trying several pretreatment conditions, not adding probe, and just adding the ProLong Diamond. At some point you will start digesting the nuclei so much that the DAPI counterstain will be going away. But the probe target you are investigating COULD be inaccessible with the pre-treatment conditions that you have tried so far.
Stacy Mazzalupo Thanks! Was the 30 min heat pre-treatment for human skin? I was more inclined to further reduce the heat pre-treatment to like 10 min, instead of increasing it.
The most likely issue is that your nucleic acid is crosslinked to much - this is normally due to excessive length in formalin. You may want a more aggressive antigen retrieval, you could heat for longer if needed - we heat for 10 mins @ 121C - as long as morphology doesn't suffer you could be okay. You could also try using a high pH buffer like Tris instead of sodium citrate. Another approach would be to use a microwave or formic acid to uncross link your samples.
Fixation time may vary for sample to sample, but your target probe will also vary by how much it is affected by fixation, its annoying but you need to try and find a good balance.
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