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Unmasking using a water bath protocol

A protocol for water bath unmasking


Water Bath Unmasking Protocol

Using a water bath at temperatures just below boiling can be a convenient method for many laboratories. Temperatures as low as 90°C can be used in conjunction with longer incubation times; the time can be shortened at 95-98°C. 

Use of a water bath below boiling should mean that loss of solution through evaporation is not an issue, but tissue sections should never be allowed to emerge from hot solution.

  1. Cut & mount sections on coated slides - VECTABOND, Poly L-Lysine or APES coatings are recommended
  2. Deparaffinize sections and rehydrate to distilled water
  3. Fill a coplin jar with unmasking solution and pre-warm to the desired temperature in a temperature controlled water bath.
  4. Add dewaxed and rehydrated sections to a slide rack and completely immerse slides in the coplin jar.
  5. Incubate for 20-40 minutes. If sections require different unmasking times, they can be added throughout this incubation, ensuring timings mean all slides finish together. 
  6. Protecting yourself from the steam, remove coplin jar with slides from the water bath and allow to cool to room temperature. DO NOT REMOVE SLIDES UNITL COOLED TO ROOM TEMPERATURE.
  7.  Wash in buffer.

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