I'm performing an in situ Hybridization protocol and have no knowledge of the expression of my candidate gene. Firstly, I will perform a qPCR in order to see when the gene of interest is expressed. I do however expect it to be expressed very early (in humans: foetal brain).
What would be an optimal positive control for this ISH experiment? I have a HER4 probe at the moment (very strongly expressed), but am sceptical of its use, because it's so easily detectable in comparison to my GOI.
Thank you for your help!
Dear Monika, a surely early gene could be ddx4 (vasa). However, you can also try a search for the best marker at the ZFIN database. There, under the Gene Expression menu, you can refine your search according to the desired stage, assay for detection, anatomical region and so on. In the resulting list of genes, you could find a marker that is maybe already available in you lab. Good luck! Natascia
A positive control probe will only tell you if your staining protocol worked or not. ISH will not provide you a quantitative read out. Easy detection means the probe binds well to your RNA target in the conditions you provide. Keep stringent temperature high to reduce background (65-70°C). Probe lengths between 500 and 1000 b penetrate easily. Until around prim-5 stage, no extra permeabilization by protease is necessary. Re-using your probe ensures that unspecific binding sites are preabsorbed (you can re-use your probe at least 5 times). Also block sufficiently (1% BSA w/v usually). Keep anti-DIG-AP concentration low and also re-use, too. Include 1% DMSO for antibody incubation helps tissue penetration, too. Washing steps including DMSO 1% helps washing out unbound Ab and reduce background. Do not use BM-purple, mix BCIP and NBT in staining buffer on your own. If all that does not work for your GOI, try optimizing your fixing regime.
Could you define very early please? I would not consider a zebrafish embryo with a fore brain to be a particularly early stage. gch2 and dct work great for stages between 20 hpf and 48 hpf.
Of course no positive control will ensure your GOI's probe is actually working, unless you see actual staining. Different probes are different worlds, if one probe works, that doesn't mean the other one will work too. Positive controls are good when you feel that there's an issue in the protocol itself and you know that X probe already worked with this protocol, so you repeat it and check if its working. Apart from that, there's no way of knowing if you're not getting staining because the gene is not there, or because the probe itself isn't working (at least in my experience).
If you want to check whether your ISH protocol works in a double reaction, I would use a reliable probe with a very localized expression, so it does not interfere with your GOI. If you expect brain expression between 24 and 48hpf, I would use cmlc2, that is expressed in the heart, leaving the brain free for your GOI.
I agree with Alejandro. Use a probe for a gene that is expressed anywhere else but the brain. Avoid genes such as ID4 and sox2 and any other genes involved in neurogenesis. Also try staining different stages using qPCR as a reference.
Thank you all for great answers! I'll go for the heart and see what happens :) thank you again!
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