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Antigen Retrieval Methods &
Controls in IHC
Dr. Usman Shams
• Formaldehyde forms methylene bridges
between proteins, which can hinder epitope
recognition by the primary antibodies.
• Two methods to remove these bridges are:
– Heat-induced epitope retrieval (HIER)
– Proteolytic-induced epitope retrieval (PIER).
• HIER is the most common approach to antigen retrieval.
• Temperature, pH and time of incubation are critical
factors that must be optimized for proper antigen
unmasking without causing morphological damage.
• Sodium citrate (pH 6) and Tris/EDTA (pH 9) buffers are
commonly used with HIER in conjunction with the heat
source (microwave oven, pressure cooker or vegetable
• Microwave oven are mostly used for this purpose.
• The time length of 15-20 minutes appear to be most
satisfactory and also cooling for 15-20 minutes.
• The 15-20 minutes are given in intervals of 5 minutes.
• The PIER approach utilizes the enzymatic activity of
pronase, pepsin, ficin, trypsin or proteinase K to partially
digest proteins to unmask the antibody epitopes.
• The efficacy of using PIER is dependent upon enzyme
concentration and incubation time.
• Antigen retrieval can be performed using either HIER or
PIER, or through a combination of both approaches.
• Frozen tissue sections do not need an antigen retrieval
step. Once mounted on APES coated slides, they are
best kept at -80°C until needed. When required, allow
the slides to warm at room temperature for 5 minutes,
then acetone fix for 5 minutes followed by a PBS or TBS
rinse. Afterwards, continue with the immunohisto-
chemical staining protocol.
• Williams and coworkers137 investigated the effect of
tissue preparation on IHC staining by using tonsil tissues
that were subjected to variations in fixation, processing,
section preparation, and storage. They demonstrated
that the microwave AR technique ameliorated the
problems resulting from variations in fixation, processing,
and section preparation. They reported that 10% neutral
buffered formalin, 10% zinc formalin, and 10% formal
saline gave the most consistent results overall and
showed excellent antigen preservation.
• Fraenkel-Conrat and coworkers, who documented that
the chemical reactions that occur between protein and
formalin may be reversed, at least in part by high-
temperature heating or strong alkaline hydrolysis. They
also noted that significant denaturation of unfixed purified
proteins occurred at temperature ranges of 70° to 90°C,
whereas similar temperatures had virtually no adverse
effect on formalin-fixed proteins (i.e., formalin-fixed
proteins are more heat stable).
Special controls must be run in order to test the
protocols and foe the specificity of the antibody being
Two types of controls are used
1. Positive controls
2. Negative controls
1. POSITIVE CONTROLS
It is taken from the known positive case.
If the positive control tissue showed negative staining ,
the protocol and procedure need to be checked until a
good positive staining is obtained.
2. NEGATIVE CONROLS
Negative control is test for the specificity of the antibody.
First the negative staining must be shown in the
omission of the primary antibody.