Tips for Step 20: Mount Coverslip

Overview

Mounting media are essential when long term storage of slides is required (for instance when the slides are to be stored for future reference purposes). They enable permanent adherence of the coverslip to the slide, thereby protecting the tissue specimen from damage while simultaneously adding contrast during microscopy.

Mounting media categories:

1. Aqueous/water-based mounting media (hydrophilic; examples of aqueous mounting media include glycerine-glycerol and glycerine jelly) (Ravikumar et al. 2014)


2. Organic-solvent based mounting media (hydrophobic; examples of organic solvent based media include Euparal and Canada Balsam) (Ravikumar et al. 2014)

Mounting media can be further subcategorized into solidifying media or those that stay liquid (Microbehunter.com no date). In general, organic-solvent based media solidify while water-based ones remain liquid.

Factors to Consider

• Aqueous-mounting media are compatible with both fluorescent and enzymatic labels (Ravikumar et al. 2014). It is important to confirm that the medium is clear before application as cloudiness is a possible indication of bacterial or fungal growth (Kiernan no date). To mitigate the risk of contamination, ensure that the medium contains bacteriostatic agents (Ravikumar et al. 2014)


• Organic mounting media should exclusively be used for mounting chromogenic IHC slides (Ravikumar et al. 2014).  However, even for these slides, exemptions exist as some precipitates (for example by reactions with AEC) are soluble in organic mounting media (Howard and Kaser 2014). For these types of precipitates and substrates aqueous mounting media should also be used


• For wide-field microscopy, mounting media that solidifies should be used (North 2006)


• To visualize 3D structures or to obtain 3D tissue information, mounting media that remain liquid are required (North 2006). When using this type of mounting media, the coverslip edges must be sealed with nail polish to avoid leaks (North 2006)


• Slides should be stored in the dark to prevent photobleaching/fading. The risk of photobleaching may be further reduced by selecting mounting media containing antifade reagents (North 2006). As certain fluorophores are incompatible with antifade reagents, it is important to first confirm suitability before using this type of mounting media

Related Resources

• IHC application page
• IHC protocols
• IHC-paraffin tested antibodies
• “Mastering IHC experiments webinar”
• Immunohistochemistry - Paraffin Tips & Tricks

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