I am embedding and sectioning mouse sciatic nerves for immunofluorescence and something very strange is happening. There don't appear to be any bubbles at first glance under the scope, but after several seconds of exposure small bubbles seem to be emerging from the sectioned tissue and the grow in size rapidly. The effect is much worse at higher power and under shorter wavelength light (presumably more energy=more heating=more bubble expansion). My question is: where are these bubbles coming from and how can I prevent them from forming? There are not any bubbles visible after coverslipping, so they must be micro bubbles within the sectioned tissue itself (or just surrounding it) that grow rapidly when heated by the fluorescent light. There are tons of them, not just a few, and they completely obscure my field, making it almost impossible to capture good images. I greatly appreciate any suggestions or ideas. Thanks.
My protocol is as follows:
1. Sacrifice animal by CO2 asphyxiation
2. Remove sciatic nerves, immerse in 1%PFA x 10 min, then PBS x 10 min
3. Cryoprotect in 30% sucrose overnight at 4 degrees.
4. Embed in OCT, flash freeze using cooled isopentane over dry ice.
5. Section at 10 um on cryostat, let sections dry on slides overnight at RT
6. Wash slides in PBS 3x5 min
7. Block in 10% normal serum in 1%BSA/.25% Triton X-100 x 1 hr at RT
8. Incubate in primary AB diluted in BSA/Triton X at 4 degrees overnight.
9. Wash in PBS 3x5 min
10. Incubate in secondary AB diluted in BSA/Triton X at RT x 2 hrs
11. Wash PBS 3x5 min
12. Incubate 1:5000 Hoescht nuclear stain x 5 min
13. Wash PBS 3x5 min, distilled H20 3x5 min.
14. Coverslip on slides using Fluroshield mounting medium (careful not to use too much or introduce bubbles), let dry overnight.
15. Seal slides with nail polish, image on fluorescence microscope
Hi, What tissue are you using? Some time in tissues like breast, fat globules comes out and look like air bubble. Try using a different mounting media
Thanks for your reply. I'm using sciatic nerves. These are definitely air bubbles that I'm seeing and not fat bubbles. I can try a different mounting medium, but I've been using the same stuff for a while without running into this problem.
I don't know how helpful this will be, but how tightly adherent are your sections to the slide? Could there be small bubbles trapped between your section and the slide that get liberated when you view the specimen? We have sometimes encountered bubbles emerging from underneath frozen tissue sections as we view them, but we haven't had the problem nearly to the extent you describe.
How old is your mounting medium? Is it possible that the mounting medium has gone off and is reacting to the intense light exposure.
The mounting medium is relatively new and other people in the lab have been using it without issue. It's possible that the sections are not adhered tightly to the slides and air is trapped underneath. I dry the sections onto the slides overnight to improve adherence and I am using slides pre-treated with a tissue adhesive so I'm not sure what else I can do to prevent that. Thanks for your reply.
When tissue is embedded in OCT, be careful to not have any air bubble in OCT. They can affect the tissue attachment to slide when sectioning which can lead to cracks or bubbles due to air pocket in sections.
An unusual problem. A few suggestions-
Do the bubbles occur if you just section an empty block of OCT, dry the sections on slides, rehydrate and then mount in your mountant and image on the microscope? This would identify if the bubbles come from the tissue or the embedding/mounting media.
Consider drying slides in a vacuum dessicator (step 5). Keep the sections frozen to the slides, in a cold rack in the cryostat. Quickly transfer frozen rack to dessicator, cover and turn on vacuum. This makes the slides dry very quickly, removing all air and water.
Thanks for your ideas. I will try cutting a block that don't have tissue, but I doubt I will see bubbles since they are all so closely localized to tissue in the other samples. The vacuum desiccator is a great idea, I will look around our department to see if I can find one to use. Hopefully I can figure it out! Take care, Brendan.
I am having the exact same problem. It seems to be somewhat random as some slides have the issue and other slides (different sample) processed at the same time are fine. Did you ever figure out the cause? Thanks!