There are two main categories: Hardening and Non-hardening mounting media
- Hardening media solidify, permanently sealing the coverslip and protecting the specimen for long-term storage
- Non-hardening media remain liquid or semi-liquid, allowing coverslips to be removed if needed
Additionally a sealant can be used to hold the coverslip in place and avoid drying. This is required for non-hardeiing mounting media and optional for hardening ones.
- TwinSil: This is the recommended sealant. It is a silicone based two conponents sealant. Mix the two component A and B in equal parts and apply around the coverslip to seal it to the slide. Pros: Easy to prepare, stable and chemically inert, does not react with ethanol, immesion oil or other solvent, does not damage objectives. Cons: Viscous (more difficult to apply), lower adherence to the glass. Apprently TwinSil has been renamed Eco-Sil with with 4 types Regular, Speed, Soft, Extrahart with different setting time and hardness.
Picodent_Silicones.pdf from https://www.picodent.de/ - Nail polish: Despite being popular, nail polish is not as good as TwinSil. Pros: Easy to prepare, cheap, easy to apply, dries quickly. Cons: Chemically active it can react with ethanol, immesion oil or other solvents. Does not dry completely at 4C which cause a risk of damaging the objectives. Wide diversity of Nail polish
- VaLaP: A mix of Vaseline, Lanolin and Paraffin): Classic sealant in microscopy. Pros: cheap and easy to prepare. Flexible and reversible. Cons: Needs to be applied at warm temperature. Can be difficult to apply on small coverslips. Not recommended when using oil objectives since the oil can be contaminated.
Many fluorophores (e.g., FITC, Cy3) are prone to photobleaching. Mounting media often contain antifade agents to slow down bleaching. The most populat is DABCO stands for 1,4-Diazabicyclo[2.2.2]octane. Available at Sigma https://www.sigmaaldrich.com/CA/en/product/sial/d27802 at 1% or 2% in the mounting media.
Antifade agent → protects fluorophores from photobleaching caused by reactive oxygen species (ROS) generated during excitation.
Free radical scavenger → reduces singlet oxygen and other reactive species that damage fluorescent dyes.
Improves signal stability → allows longer imaging times and repeated scans, especially important for sensitive fluorophores like FITC, TRITC, Cy3.
This is really important: To minimize spherical aberrations and improve resolution, the medium’s Refractive Index should match the glass coverslip (~1.515) and the immersion oil (~1.518).
