Bubbles forming during hybridization in fresh frozen tissue

Meshel8 · **Meshel8**

I'm struggling with an annoyance, which is really starting to screw up my ability to get good data.

I do ISH with DIG RNA probes. The probes are diluted in hybridization buffer, denatured, returned to ice, then applied to slides of fresh frozen mouse brain sections, 15 microns thick.

Hybridization is done inside a humidified box in a chamber at 56 degrees overnight. I use hybrislips by Grace Biolabs as an alternative to parafilm or glass slides for covering the tissue. The probe goes on nicely and the hybrislips lay down beautifully, but bubbles form under the hybrislips during hybridization. So there are big areas of tissue, like crop circles or something, where there is no hybridization. After the color reaction, these areas are perfect white bubbles that have clearly not had contact with the probe.

These bubbles are a huge problem. It's a crap shoot every time, whether or not bubbles will form in the areas most important to our lab.

I've tried several things to make this problem go away: I switched to Hybriwells, which, though sealed, had the same problem as the hybriwells. I conclude from this that the bubbles are coming from the hyb buffer or the tissue.

I have tried warming the probe and hyb buffer prior to putting it on the slides. I have tried removing all residual buffer by letting the slides dry briefly before putting on the hyb buffer. I have put the hyb buffer on the slides, placed them in the humidity chamber for 15 minutes or so and then put on the coverslips. This last has been the most successful; although bubbles still do form unpredictably, there seem to be fewer of them. It is quite difficult though logistically: trying to figure out how to move the slide with the hyb buffer on it out of the hyb chamber & cover it with a hybrislip without having it cool, thus producing bubbles when put back in the chamber.

Has anyone else struggled with this? I have searched everywhere on the web that I can think of. Please send any and all suggestions.

THANK YOU.

gula · **gula**

Is there a mechanism for sealing at this special slides?

I use usual glassslides with usual glass coverslips and seal it at the edges with rubber cement. After drying the sealing avoids loosing liquid during heating.

gula

Meshel8 · **Meshel8**

Hi Gula, Thanks for your response. I don't seal the coverslips on the slides. We use a humidity chamber (tupperware box) to keep the slides from drying out. We tried using hybriwells, which seal the tissue and hyb buffer from using sealing sides and ports that you cover with seals. Even in this situation, bubbles formed and were problematic.

I have read a few things since I posted that indicate that the composition of the box buffer may be important. I usually just use water or PBS to wet the paper towels in the humidity box, but several protocols use a solution of 50% formamide and 4X SSC to control the osmolarity and humidity. I wonder if this could help prevent the formation of bubbles. I am going to try this in my next round of ISH to see if it helps.

Any/all suggestions or comments are very much appreciated.
Thanks,
Michelle

xylenefumes · **xylenefumes**

How about heating the hyb solution and probe to a temp 5-10 deg higher than your hyb temp, and setting the chamber temp to that higher temp, placing the covers on the slides, putting all in the chamber, then bringing the chamber temp down to the hub temp?

nickd · **nickd**

My lab had the same issues back in the day causing a lot of data to fail unnecessarily. The trick to clean bubble free data was degassing (in a vacuum chamber) a subset of reagents used during the ISH process. Most reagents need about 30-45 minutes in the vacuum chamber to pull out the dissolved gases causing the bubbles. We ended up having to degas about 10 of our reagents used in the ISH protocol to ensure that we'll end up with bubble free data.

Good luck!

Immunohistochemistry	In Situ Hybridization	Immunostaining
Immunohistochemistry Images	Tissue staining	Pathology
Tumor Markers	IHC Reagents	Tissue Micro Array
Immunofluorescence Staining	Positive Control Slide	Polyclonal Antibody
Monoclonal Antibody	IHC Staining Protocol	In Situ Hybridization Images
Immunohistochemistry Protocol	Immunofluorescence	Immunohistochemical Staining
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