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

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