I am having difficulties with the in situs. I use embryonic mouse tissue (days 9.5 to 12.5) that have been fixed 2h to ON (depending on the tissue and what I'm trying to do with them). The issue I'm running into is that there is a gradient of staining within a slide, from one border to the other. I have seen that tilting the oven during the hybridization part (trying to make it 100% flat is impossible) changes the results, but I still can't get rid of the problem. So it definetly has to do with either the distribution of the probe or the fact that the hybridization mix gets dryer on one side of the slide than the other; even though they never actually get dry because the glass coverslips are super easy to get off my slides.
Anoyne has encountered, fought this problem and won the battle?
Hi Daniela,
Since you mentioned, that "tilting the [hyb] - oven" changes the result, is it possible, that the slides are not level during the steps performed in the humidified chamber ?
Maybe it helps to increase the volumes of the solutions, for those steps, which are performed on the slides.
Best Simone
I don't know, if this method is possible with your slides. I work with 3 µm tissue-sections of FFPET. While hybridization the section is covered with a coverslip and sealed with rubbergum. So hybrization-solution is spread equally and cannot dry out. I work with a hybridizer (hot plate with lid).
Hi Daniela,
We encountered the same problem. First of all, when cryosectioning, we try to avoid putting sections at the side of the slide, by systematically placing them in the middle (if possible). We also bought a special cover slip with lifted edges so that the hybridisation solution with probe can spread homogeneously, and no gradient towards the side of the glass develops. These cover glasses are called HybriSlip from Sigma-Aldrich. Unfortunately, still some variation in staining is still present, and I think it can never be completely avoided, but this may help. Good luck.
Hello everyone and thanks for your answers!
Pieter, I'm both relieved and sad to know that you've ran across the same issue. I'm going to look into these coverslips. Do you think they really help? Or is it more avoiding the edges than the actual coverslips?
Gudrun, what is this rubbergum you talk about? Do you have a catalog number/brand I can look into?
Thanks everyone for your time. We're also going to buy a new oven since ours is kind of old already.
Hi Daniela,
We do believe the cover slips help, and use them consistently. But indeed it is difficult to judge how much they improve our staining, because the ISH signal itself varies a lot. Avoiding the edges is maybe the first thing you can try. We just systematically noticed the ISH signal to be fainter on the edges. I always see the staining as a proces of optimisation, and combination of the cover slips and avoiding edges is one part of this process.
And certainly a new oven is important. Temperature can be unstable or vary inside the oven, and reduce quality of the staining. We always check by putting a thermometer in the oven.
Best of luck!
The rubbergum is a kind of glue, that is eg. used to stick photos in an album. It can be removed completely again. I use a product called Fixogum (in Austria).
http://www.marabu.com/creative/produkt/ansicht/?pid=290117000
Put the probe on the section, cover it with the coverslip without any airbubbles (that would expand in the heat). Cover the edges of the coverslip with a big amount of rubbergum. The edges have to be sealed completely! Let the glue dry until it is transparent and covers the edges with a thin film.
After hybridization lift a corner of the rubbergum with a scalpel and peel it carefully off again. The coverslip can be washed off in a buffer or can be lifted carefully also with the scalpel.
We use coverslips 15x15 mm.
Look at this instruction manual of Dako. It shows the usage of rubbergum.
http://www.dako.com/at/29052_her2_iqfish_stepbystep_procedure.pdf
Thank you very much to both for you. These are two different approaches i deffinetly need to try.
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