In the field of electron microscopy, achieving high-quality images of samples is crucial. One key step in this process is fixation, which stabilizes samples and prevents them from degrading during processing. Tannic acid has emerged as a popular choice for fixation, thanks to its ability to preserve cellular structures and enhance contrast. In this blog, we will explore the benefits of tannic acid fixation and outline a protocol for its use.
Tannic acid is a naturally occurring compound found in various plant species, including oak bark and tea leaves. It is a polyphenol with a high affinity for proteins, making it an effective cross-linking agent for biological samples. Tannic acid has been shown to preserve cellular membranes, cytoskeleton, and extracellular matrix, making it a useful tool for studying various tissues and cell types. Additionally, tannic acid has the ability to enhance contrast in electron microscopy, making it easier to distinguish between different cellular structures.
To use tannic acid for fixation, a specific protocol must be followed. The Marclab for Connectomics provides a detailed protocol that we have summarized below:
1. Prepare the fixative solution by mixing 2.5% glutaraldehyde and 1% paraformaldehyde (or 0.1% glutaraldehyde and 4% paraformaldehyde) in 0.1 M cacodylate buffer. Add 3% sucrose and 1mM magnesium to the solution.
2. Fix the sample with the desired fixative by immersion or intraocular injection.
3. Follow with 30 minutes of fixation on ice.
4. Rinse the sample well three times for 10 minutes each in 0.1 M cacodylate buffer on ice.
5. Incubate the sample for 40 minutes in tannic acid on ice.
6. Rinse the sample well two times for 10 minutes each in maleate buffer on ice.
7. Incubate the sample for 40 minutes in dark uranyl acetate on ice.
8. Rinse the sample well two times for 10 minutes each in maleate buffer on ice.
9. Wash the sample for 10 minutes in 75% ethanol on ice.
10. Optional: incubate the sample for 15 minutes in para-phenylenediamine on ice to enhance membrane contrast.
11. Rinse the sample three times for 10 minutes each in 75% ethanol on ice (optional).
12. Rinse the sample for 10 minutes in 85% ethanol on ice.
13. Rinse the sample for 10 minutes in 95% ethanol on ice.
14. Rinse the sample two times for 10 minutes each in 100% ethanol on ice.
15. Rinse the sample for 10 minutes in 100% acetone on ice.
16. Rinse the sample for 10 minutes in 100% acetone at room temperature.
17. Incubate the sample for 60 minutes in 50% resin in acetone.
18. Incubate the sample for 4-20 hours in 75% resin in acetone.
19. Incubate the sample for 60 minutes in 100% resin.
20. Embed the sample in fresh resin in molds and cure at 60-65°C overnight.
Following this protocol, samples can be prepared for electron microscopy with improved contrast and preservation of structures. Tannic acid fixation is a powerful tool for studying various tissues and cell types, and it has been used in numerous research studies.
In conclusion, tannic acid fixation is a valuable technique for achieving high-quality images in electron microscopy. Its ability to preserve cellular structures and enhance contrast makes it a popular choice for many researchers. By following the protocol outlined here, researchers can create samples that are optimized for electron microscopy and achieve the best possible results for their research.