Protocol for HIER/HTAU in a microwave for IHC
There are numerous techniques to unmask antigens, mostly utilizing pressure cookers, however some labs may prefer to avoid more regulations by avoiding pressurized vessels. High temperature antigen unmasking/ epitope retrieval can be done simply in a microwave, with the right solution in a coplin jar covered with clingfilm, although alternative vessels can be used.
Points to note before you start:-
Microwave ovens can have hotspots – these can induce artifacts, but there’s an easy way to get to know your microwave. You can spot them by putting a slice of buttered bread in your microwave for a few seconds & see where the butter melted. Try to avoid placing the vessel on these spots.
Ensure slides have space around them so that fluid does not get trapped in pockets – if fluid can’t circulate the solution will not heat up evenly and unmasking won’t be consistent. If the slides are too tight it can also lead to flash drying type artifacts.
ALWAYS allow the slides to cool in fluid to prevent flash drying. If you need to get at your slides quickly, put your coplin jar with your slides and fluid into the sink and add water slowly to replace the hot solution until the slides are adequately cooled. Don’t be tempted to throw away the hot solution then add water to cool the slides.
High temperature unmasking techniques can be pretty tough on tissue sections – it is strongly recommended to use subbed slides with some form of adhesive. Without adequate attachment sections and cells are likely to become detached from slides. (See our article:- Section preparation) If this is not possible, a water bath method of unmasking may be advisable.
- Cut & mount sections on coated slides - VECTABOND, Poly L-Lysine or APES coatings are recommended.
- Deparaffinise sections and rehydrate to distilled water.
- Slides are placed in a coplin jar containing Unmasking Solution covered with clingfilm (vented), and heated in a 650-800W microwave oven for two 5 minute cycles at full power (if the buffer evaporates after 5 minutes, top up the fluid with hot distilled water). The number of treatments in the microwave should be determined by the individual lab.
- Let the sections stand for 20 minutes in the microwave (very important)
- Wash slides in TBS buffer (pH7.6) for 5 minutes.
- Continue with immunohistochemistry protocol.
Commercially available high & low pH antigen unmasking solutions are suitable for this method. To get a quote for the products or to place an order, please email Sales@2BScientific.com.