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Mounting media is crucial a crucial component of your microscopy experiment. It has several roles:

  • Sample preservation, stabilization and protection against drying
  • Preservation of fluorescence signal against photobleaching (antifade agents)
  • Optical clarity & Resolution (refractive index (RI) matching)

Summary

  • For IHC use DPX
  • For IF use SlowFade Glass
  • To seal your coverslip use a silicone based sealant (Picodent Twinsil)

List of mounting media

NameManufacturerTypeAntifadeRefractive IndexCAD / mLCommentsLink
VectaShield PlusVector Labs / BioLynxNon-HardeningYes1.4530
https://vectorlabs.com/products/vectashield-plus-antifade/
VectaShield VibranceVector Labs / BioLynxHardeningYes1.47 (cured)33
https://vectorlabs.com/products/vectashield-vibrance-antifade-mounting-media/
VectaShieldVector Labs / BioLynxNon-HardeningYes1.453
https://vectorlabs.com/products/vectashield/
VectaShield HarsetVector Labs / BioLynxHardeningYes1.46 (cured)39
https://vectorlabs.com/products/vectashield-hardset-antifade/
Fluoromount GInvitrogenNon-HardeningYes1.4254.8
https://www.thermofisher.com/order/catalog/product/00-4958-02?SID=srch-srp-00-4958-02
SlowFade GlassInvitrogenNon-HardeningYes1.5240.0
https://www.thermofisher.com/order/catalog/product/S36917-5X2ML?SID=srch-srp-S36917-5X2ML
SlowFade DiamondInvitrogenNon-HardeningYes1.4235.3
https://www.thermofisher.com/order/catalog/product/S36972
SlowFade GoldInvitrogenNon-HardeningYes1.4231.4
https://www.thermofisher.com/order/catalog/product/S36936?SID=srch-srp-S36936
ProLong GlassInvitrogenHardeningYes1.5144.7RI reached after 60 hhttps://www.thermofisher.com/order/catalog/product/P36984?SID=srch-srp-P36984
ProLong GoldInvitrogenHardeningYes

1.46

38.2

RI reached after 48 h

https://www.thermofisher.com/order/catalog/product/P36934?SID=srch-srp-P36934

Prolong DiamondInvitrogenHardeningYes

1.46

39.7

RI reached after 48 h

https://www.thermofisher.com/order/catalog/product/P36970?SID=srch-srp-P36970

MowiolHome madeSemi-hardeningYes1.42<1.0See recipe below
EntellanMillipore SigmaHardeningNo1.496.1
https://www.sigmaaldrich.com/CA/en/product/mm/107960
Canada BalsamMillipore SigmaHardeningNo1.5254.8
https://www.sigmaaldrich.com/CA/en/product/sial/c1795
DPXMillipore SigmaHardeningNo1.524.4
https://www.sigmaaldrich.com/CA/en/product/sigma/06522

Mowiol Recipe

Ingredients (for ~10 mL stock solution):

Preparation

  • Slowly add Mowiol 4-88 to distilled water while stirring
  • Heat gently (~50–60°C) to dissolve completely (do not boil)
  • Add glycerol and mix thoroughly until totally disolved
  • Add Tris-HCl buffer to maintain pH
  • Add antifade (DABCO) and mix thoroughly until totally disolved
  • Use a 0.45 μm or 0.2 μm filter to remove undissolved particles
  • Store at 4°C, protected from light
  • Can be kept for several months.

Pro Tips

Procedure

  • Add a small drop of mounting medium on the slide
  • Place the coverslip gently to avoid bubbles
  • Let it cure at room temperature (usually overnight, in the dark)
  • Once hardened, the coverslip is permanently sealed

Tips

  • Use just enough mounting medium to cover the sample — too much will cause the coverslip to float
  • Avoid trapping air bubbles by lowering the coverslip at an angle
  • Always cure/seal in the dark to protect fluorophores.
  • Store slides flat, at 4 °C, in a box with desiccant if possible.

Relevant information about Mounting Media

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

Sealant

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.

Antifading

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.

Refractive index matching

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). 

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