I have sections of tissue on slides and looking at the bacteria in the tissue, but haven't got any results. Can anyone give me some feedback on my protocol please:
Day 1
1. Wash twice in 1X PBS for 15 minutes
2. Wash in 0.05% triton X-100 in 1X PBS for 20 minutes
3. Denature at 60C in 2x SSC/50% formamide (prehybridisation buffer) for 40 mins
4. Meanwhile place 0.5 uL of EUB338 cy3 probe in 24.5 uL of hybridisation buffer (2XSSC/50% formamide, 12% dextran sulphate) and heat to 95C for 3 mins in PCR block
5. Transfer probe immediately to ice to prevent reannealing
6. Add hybridisation buffer containing the probe, put coverslip over and seal with fixogum.
7. leave overnight at 37C.
Day 2
1. 2XSSC/50% formamide (prehybridisation buffer) for 15 minutes at 40C. Repeat once.
2. Wash twice, as above, in 2XSSC for 15 minutes at 40C
3. Wash twice in 0.1XSSC for 15 mins at 60C
4. Wash 6 times in 0.05% Tween20 in 1XPBS at RT for 15 mins each
Thank you for any feedback
Dear Caroline,
I suggest to add SSC only after the hybridisation step, since it could interfere with probe-mRNA hybridisation. First the probe should have the opportunity to bind to the mRNA, and afterwards any probe that is bound aspecifically to non-target mRNA can be washed away with SSC. I would also to wash with SSC at the same temperature (40 instead of 60°C).
Another possible problem could be the Cy3 signal that is too weak. Has this probe been used before? Is the concentration high enough? Is there any background signal noticeable? Is there enough Cy3 incoorporated in the probe? Is gene expression high? These things could altogether indicate where a possible problem might occur. If possible you could add positive controls, i.e. samples in which you know this gene is highly expressed.
Hope this helps,
Pieter
Thanks great thanks!
So you are saying miss out step 3 completely or just do that step in formamide?
The cy3 is already bound to the primer and was ordered in as standard. I was worried about the concentration of primer, the primer with probe attached is diluted to 3.2 pmol and then 0.5 ul added to the hybridisation buffer.
I was thinking of using a slide with just ecoli smeared on it to optimise the conditions.
Very helpful, thank you
Caroline
Indeed, I would suggest to do the step in the prehybridisation buffer to 'prepare' your tissue for optimal probe binding, but avoid the SSC.
The dilution of the probe is quite low, did they used it in other experiments before? It will definitely be worth it to include a positive control. Adjusting probe concentration and pretreatment of the tissue is good to start working with.
From the moment you have confirmation of a specific signal, you can adjust other steps like the SSC to optimise your protocol. Maybe your 0.1 SSC step is also too stringent so that more probe is able to bind, you could for instance only work with 2X SSC.
Hopefully it works! Good luck with trying :).
Pieter
Yes it's a standard probe but I can't find the concentration for it anywhere, I'll have another search
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