Hi ResearchGate community, how are you? I need to request your help to mitigate the generation of microbubbles detected during the slide preparation of sample metaphase chromosomal spreads. I routinely detect the quality of the stain and integrity of the mitotic chromosomes beforehand with DAPI where consistently no microbubbles have been detected.
However, after my slide preparation with my desired immunofluorescent (IF) stains I finally protect the stained chromosomes with a coverglass, mounting media, and nail polish seal. My lab uses the EMS Shield Mount with anti-fading as its mounting media. I believe this is the source of the microbubbles from a prior inquiry. To mitigate any introduction of air, we store this mounting media in the fridge (per manufacturer's directions) and securely upside down. Whenever I am ready to seal my next slide, I simply push out any initial air bubbles on a paper towel to ensure no air has been trapped when I apply 2 drops to a coverglass and proceed as previously described.
I do recognize that with decreasing volume of the stock mounting media I will have a greater chance for air to be trapped within the stock bottle hence the storage of the stock mounting media bottle upside down. However, my last attempt to seal my metaphase spreads yielded this image (attached) of a microbubble-ridden sample prep. I walk away defeated! Only after applying the mounting media do I see evidence of those microbubbles, they were not present at the DAPI quality check step.
Has anyone else overcome how to completely overcome the introduction of microbubbles in their IF slide preps? I have implemented the prior steps I read on researchgate.net to mitigate such outcomes, but I'm open to any suggestions. We are purchasing a new mounting media bottle, but having this knowledge would help preserve my samples for my next IF slide prep.
Thanks, I appreciate any time and effort you are able to provide to me.
As you said, the DAPI works well without any air bubbles. I believe, you are talking about the DAPI with FluormountG for hardening and can be stored at RT. Also, you are saying, Shield Mount with anti-fading as its mounting media might be the problem.
I suspect, the non-hardening mounting media and storage at 4C due to its nature, plus back and forth with the imaging and carrying might have introduced such artifacts (if any). Here, the amount of mounting media used is also too much.
May be you would try to stick to hardening mounting media with DAPI or other nuclear markers, which would not change or create space while you carry or image them. And may be try to use as optimal as possible, to get specific signal.
Good Luck.
Subash Chandra Malik, thank you for your response. When I conduct the quality check step, the cells at that point have only been fixed with paraformaldehyde, stained solely with DAPI, and protected by a large cover glass to mitigate any artifacts. I do not use mounting media at this step. Slides are briefly stored at 4C until I can image my slides.
Only when I add the mounting media later in the protocol do I detect the micro bubbles. Proper storage conditions for the mounting media is 2-8C per the manufacture’s instruction and use 2-3 drops of mounting media per slide are recommended.
As for the hardening of the mounting media, I’ll need to contact the technical support because I cannot find information about this on the company's website.
Unfortunately, I have not worked with such detailed structures within nuclei, so I cannot comment on sample morphology.
But if you suspect it is due to the presence of small air bubbles in an almost empty mounting medium bottle, maybe you could try squeezing some mounting medium into an eppendorf tube, and centrifuge it to remove air bubbles? Then use a pipette to transfer the mounting medium on the slides just up to the first stop.
We use this handy little technique to make the most out of a bottle of mounting medium. After that you can even continue to store the tube at 4C. Hope this helps!
Jen C., thank you for your response. This seems very feasible and helpful! Yes, I believe this is exactly what I need to do. Thanks!
Subash Chandra Malik, I was able to find on the manufacturer's website that our mounting media is 'non-permanent' to enable ease with any additional slide staining. Thanks for that consideration.
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