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Antigen Unmasking

Also known as epitope retrieval, antigen unmasking helps make your target available to the primary antigen to bind, particularly when formalin fixation has caused conformational changes in the protein structure.

Antigen Retrieval

The art of antigen retrieval in fixed tissues can be difficult to nail down to a ‘one size fits all’ – partly down to possible variations in fixation between different laboratories, and the particulars of the antibody. 

Although predominantly of concern for detection of protein targets in FFPE tissues, there are occasions when antigen unmasking can benefit the results in seemingly unnecessary circumstances, for instance, when lectins are used to detect carbohydrates of interest that may have been sequestered by conformational changes in the proteins around them, even though the carbohydrate binding site of interest is unaffected itself by the fixation process. (Lectin Histochemistry, a concise practical handbook). It is therefore worth investigating whether an unmasking protocol is of value whenever a new protocol is being worked up, and which is most effective for the target in question. If acetone or neutral buffered formalin (NBF) has been required to preserve soluble antigens in frozen sections there may be cross linking that can be reversed by an unmasking protocol.1  

Commercially prepared antibodies should be supplied with the manufacturer's guidance as to the capabilities of the antibody for use on sections fixed in different ways, and the optimal unmasking protocols (if even required). If presented with an in house antibody, these parameters may be unknown and will need to be determined experimentally 2

Unmasking falls into 2 broad groups, digestion using enzymes, and high temperature antigen unmasking/heat induced epitope retrieval (HTAU/ HIER). With both methods, it is possible to over unmask and effectively destroy the tissue, which may lead to inappropriate variations in the staining so proper controls should be run to ensure that the staining still conforms to the expected staining pattern.

Unmasking antigens can often expose the sections to a lot of turbulence in the surrounding fluid which means that sections are more likely to become detached from the slide. It is strongly recommended to use slides with a good section adhesive to minimise section loss in any unmasking protocol. Some methods described here have less agitation than others, so this may affect the preferred method of unmasking.

Enzyme Digestion

Proteolytic digestion of the covalent bonds formed during formalin fixation can help the primary antibody reach its intended target.      

Key enzymes used for unmasking are Trypsin, Protease, Pepsin, Neuraminidase, Proteinase K and Pronase.  Each of these enzymes cleaves proteins at different protein motifs, so may have marked effects on the results.

Enzyme digestion is relatively straightforward, the enzyme solution is prepared in solution suitable for the enzyme’s functions and applied to the section. Key variables are based on the enzyme kinetics, namely temperature, dilution and time, which allows for a fairly fine control on the digestion. Batches of enzymes may vary, so new lots should be assessed and optimised.

When making enzyme solutions for use, it is recommended to prepare them just before use in pre-warmed buffer, to help control the temperature variable.

For the less expensive enzymes, immersion in the enzyme solution may offer the most even & uniform digestion compared to applying a small amount on each slide.

Ready to use cocktails of enzymes are available, such as the Zytomed Fast Enzyme – offering advantages of 5 minute digestions, and solutions stabilized to grab & go. Do ensure that there is consistency in enzyme temperature when used – incubation times may vary if the reagent is either at room temperature or straight out the fridge.

PIER Methods

High Temperature Antigen Unmasking/ Epitope Retrieval

Without & with Antigen Unmasking
Without Antigen Unmasking.
Without & with Antigen Unmasking
With Antigen Unmasking. Cyclin D1 staining on lymph node, and unmasking with Vector Laboratories’ H-3301, High pH Unmasking Solution.

There are many discussions as to the action of heat mediated unmasking protocols – what has the most effect on the unmasking properties, is it the pH, chelating properties, method of heating etc? As such there are several solutions to choose from, and this may need to be determined experimentally for each antigen.

The use of EDTA in the buffer at either pH 6 or pH 9 as a chelating agent seems to offer a ubiquitous improvement over the buffer pH alone.

Where antigens are expressed in low levels the high pH 9.0 retrieval solutions seem to be more effective, whilst citrate buffers at pH 6 are generally good all-rounders and less likely to generate false positives by over unmasking.

These solutions can be used in a variety of heating methods which have their various pros and cons.

Controlled settings

Water bath



Pressure cooker

Automated device


(up to 100oC)

(100oC only)

(up to 125oC)

Time of heating

Cooling down

Temperature: Microwaves can have uneven heating due to hot spots, either avoiding these places on the microwave plate, or allowing for good circulation of unmasking solutions can minimise this effect. For automated devices, maximal temperature can differ in each automated device.

Time of heating: In water bath, steamer and microwave the starting time of slides reaching the desired temperature remains an estimate and varies with the size of the slide container. This can be standardised, and by pre-warming the unmasking solution before the addition of slides.

For all devices, care should be taken to ensure that adequate buffer is added to completely cover the sections at all times. Sections should always be arranged to allow fluid to move freely around the slides. Inhibition of convection currents may mean that solutions have temperature variation which can lead to uneven unmasking.

Some sections may require a shorter unmasking time than others, and use of a microwave, water bath or steamer may allow addition of these sections to the protocol during the unmasking timings, not possible in a pressurised vessel! Sections requiring shorter times should be added at suitable timings to allow all the sections to be completed at the same time. 

Sections should NEVER be taken out of hot buffer as flash drying artefacts may occur. Sections must be cooled in solution – at an appropriate point, cooler water can be added to expedite the cooling process if required to ensure slides are adequately cooled.

Unmasking Solutions

Unmasking Protocols

Mixed Unmasking protocols

Some antibodies require a quick proteolytic incubation followed by high temperature unmasking too. E.g. LP34 anti-cytokeratin 5/6/18. This is normally indicated on any data sheet.

Dewaxing and unmasking automated modules.

Commercial dewaxing and unmasking modules offer convenience in that the FFPE sections can be put through this module directly from mounting and be ready for blocking and application of the primary antibody. However, although they offer a level of standardization, it reduces the flexibility often required in a research setting. Some antigens may not require unmasking – so this cannot replace the need for the series of graded alcohols in the lab.  Pre-treatment modules can also ‘finish’ whilst slides are still hot – so the temperature of slides on removal should still be considered to avoid flash drying.

All In One Solutions