IHC deparaffinization protocol

Procedure for deparaffinization of paraffin-embedded sections before staining. 

Print this protocol.

Before proceeding with the IHC staining protocol, the sdes must be deparaffinized and rehydrated. Incomplete removal of paraffin can lead to poor staining of the section. 

Materials and reagents

100% ethanol
95% ethanol
70% ethanol
50% ethanol


Place the slides in a rack, and perform the following washes using a Coplins jar:

SolutionIncubation time
Xylene3 min
Xylene3 min
1:1 Xylene:100% Ethanol3 min
100% Ethanol3 min
100% Ethanol3 min
95% Ethanol3 min
70% Ethanol3 min
50% Ethanol3 min

Once the slides have been washed in the above sequence, place slides in running cold tap water to rinse off ethanol.

Keep the slides in the tap water until ready to perform antigen retrieval. At no time from this point onwards should the slides be allowed to dry. Drying out will cause non-specific antibody binding and therefore high background staining.