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IHC Tip 5: Mounting media an underestimated success factor for bright stainings

Bio-Rad
Jun 02, 2015

Mounting media are essential for making permanent slides and assist in making the coverslip adhere to the slide. Mounting has the purpose of protecting the specimen from damage while adding contrast during microscopy. Two types of mounting media exist:

  1. Aqueous (hydrophilic; examples of aqueous mounting media are glycerol and glycerol jelly)
  2. Organic solvent based (hydrophobic; examples of organic solvent based media include Euparal and Canada Balsam)

Mounting media can be further differentiated into media that solidify or stay liquid (1). In general organic solvent based media solidify, while aqueous ones remain in a liquid state.

Factors to consider:

  • Do not use organic mounting media for fluorescent labels. 
  • When using organic mounting media care should be taken as some precipitates, formed as the result of the chromogen substrate reaction, are alcohol soluble. One of those examples is the red precipitate formed as the result of the reaction of horseradish peroxidase (HRP) and 3-Amino-9-Ethylcarbazole (AEC) (2).
  • For wide-field microscopy mounting media that solidify should be used.
  • For 3D imaging mounting media that stay liquid have to be used (3). When using mounting media that remain liquid, the sides must be sealed with nail polish to prevent the medium from leaking.
  • To prevent photobleaching (a process in which the chemical destruction of a fluorochrome results in the loss of fluorescence) slides should be stored in the dark.
  • Further bleaching can be prevented by using mounting media containing antifade reagents. However as not every fluorochrome is compatible with every antifade reagent; we recommended to check the manufacturer’s datasheet prior to use.

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References:

  • (1) Kim, O., MicrobeHunter Microscopy Magazine, An overview of mounting media for microscopy.  Available here (Accessed: December 02, 2014).
  • (2) Renshaw, S. (editor) (2007) Imunohistochemistry. Methods Express Scion Publishing.
  • (3) North, A.J. (2006) Seeing is believing? A beginners' guide to practical pitfalls in image acquisition. JCB vol. 17 no. 1 9-18.

 

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