Mounting Media for Immunohistochemistry
The purpose of the mounting media is to help preserve the staining and aid the passage of light through the slide and section with minimal refraction or distortion of the light beams passing through.
Although there are a number of recipes for mounting media for general histology, it is heart-breaking to see staining, that has taken hours to complete, vanish before your eyes when an incompatible mounting media is used in immunohistochemistry! If mounted correctly, some stained sections can still be viewed decades after they were first prepared.
The mounting media and protocol needs to be selected based on the requirements of the substrate(s) and counterstain used. Mounting sections successfully can take a little practice. Air bubbles under the coverslip can expand with time and impede imaging, so it's worth taking the time to have another go if you can see there are bubbles.
The refractive index (RI) of glass is 1.51, and tissue sections are usually 1.38-1.46. Ideally mounting media should have an RI close to that of glass for the least distortion of light, but between the RI of the tissue and glass is often adequate.
Caution! Although non-aqueous mountants are referred to by some companies as ‘permanent’ this is sometimes also used by other companies to describe their hardening aqueous mountants.
These mounting media are solvent/ resinous based and are required for substrates and counterstains that are soluble in water, such as DAB + nickel, or Methyl Green. These mountants normally set and generally do not require any sealing agent round coverslips to immobilise them. The RI will change as it dries, but should be stable once set.
As water is immiscible with these solutions, sections are passed through alcohols to remove water before mounting. For most this is a full dehydration and clearing protocol, usually the reverse of the hydration protocol used for paraffin sections before staining. An exception is VectaMount Express.
Xylene was a common base for the most mounting media, including DPX and Canada Balsam, however the toxic effects of this solvent have led some commercial companies to seek alternative formulations, although these media should still be handled with appropriate caution.
VectaMount Express H-5700-60 RI 1.49
Long term storage – (New product, so data only at 18 months -so far). Advantage is that sections only need 2x1 minute washed in isopropyl alcohol (99%+) before application of the mounting media. Thicker sections will need longer washes. Compatibility known with Vector’s substrates and H&E stains.
VectaMount H-5000-60 RI 1.49
Long term storage (Decades). This mounting media has been around for over 20 years, originally developed with a citrus oil base to improve crispness of alkaline phosphatase substrates compared to those mounted in DPX.
Mount quick DAI-DM01-1 Cosmo RI 1.41
Short term storage only, photograph ASAP. * Not BCIP/NBT Compatible
For substrates such as AEC that are soluble in alcohol, an aqueous mountant is required. They can be particularly useful when speed is of the essence as the dehydration protocol is not required, however substrates like DAB, that could be mounted aqueously or non-aqueously, will tend to stay vibrant longest in the non-aqueous media.
Aqueous mountants can continue to dry out over time so edges of the coverslip can be sealed with rubber cement or nail varnish to slow or prevent artefacts of drying mounting media forming. The drying rate of hardening media is lessened by the dried media around the edges, but it can still continue, so sealing the edges may still be advisable if sections are to be stored for a longer time.
Aqueous Mount ZY-AMT060 / ZY-AMT030 Zytomed
VectaMount AQ H-5501-60 Vector Laboratories
Mount Quick “Aqueous” DAI-DM-02 Cosmo – Not for long term storage
If air bubbles or other mounting media artefacts develop over time, it is often possible to soak off the coverslip and re-mount the section. For aqueous media, PBS can be a good choice, or the clearing agent for the non-aqueous media. If the section has been mounted for a while, it may need a longer soak. Test with a spare section if possible to check there are no adverse effects before trying this with a larger batch, or particularly important sections!
If non-aqueous mountants look cloudy, it can be that the dehydration protocol was insufficient. Soak off the coverslip, and take back down the alcohol gradient to water. Check if alcohol solutions need changing (their % alcohol changes over time with water carry over, and water extracted from tissues) Run the dehydration again & re-mount.