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1. ANTIGEN RETRIEVAL
By:
1. Siti Swaprziah Bt Muhyiddin 03-200904-00273
2. Siti Nadiyah Bt Othman 03-200904-00387
3. Sunarti Bt Mansur 03-200904-00199
4. Syloveria Binti 03-200904-00201
5. Nurhanisa Bt Gulamu 03-200904-00130
6. Suierina Lawansing 03-200904-00226
7. Randy B. Gubud 03-200904-00264
DMLT 0904 Group 3
MLTA 3812
ANATOMY PATHOLOGY 4
PRESENTING:-

2. Objectives:
 Define antigen retrieval
 Describe the principle of Proteolytic enzyme
digestion method and its procedure
 Describe the principle of Heat induced
epitope retrieval method and its procedure
 State the staining procedure
 LSAB-peroxidase

3. Content:
 Definition of antigen retrieval
 Epitope
 Masking of antigen / epitope
 How to retrieve masked antigen
 Antigen retrieval technique categories
 PIER
 HIER
 Combination of HIER & Enzyme
 Procedure of PIER, HIER+Enzyme and HIER
 LSAB staining

4. Antigen Retrieval
 Method performed to expose or retrieve
antigens which have become masked by
series of events.
 Technique in which the masking of an epitope
is reversed and epitope-antibody binding is
restored.

5. Epitope

6. Masking of antigen/epitope:
 Happen when the epitope is covered and can
no longer bind to the primary antibody.
 caused by:
 Fixation can alter protein biochemistry
 cross-linking of amino acids within the epitope
 Cross-linking unrelated peptides at an epitope
 Altering the conformation of an epitope
 Altering the electrostatic charge of the antigen

7. How to retrieve the masked
antigen?
 Pretreatment with the antigen retrieval
reagent
 Break the protein cross-links formed by
formalin fixation
 Thus uncover hidden antigenic sites

8. Antigen Retrieval Techniques
 Antigen RetrievalTechniques basically
divided into 3 categories:
 Heat Induced Epitope Retrieval (HIER)
 Proteolytic Induced Epitope Retrieval (PIER)
 Combination of Heat Mediated and Proteolytic
Enzyme Method

9. Protease-induced Epitope
Retrieval (PIER)
 Enzyme used:
 Protease
 Trypsin
 Pepsin
 Principle:
 Enzyme will breaks down formalin cross-linking
and hence the antigenic sites for antibodies
binding are uncovered

10. Disadvantages of PIER
 May destroying tissue morphology
 May also destroy epitope of the antigen
 Finally leading to false negative results

11. key factors to be considered
when performing PIER
 Concentration of enzyme is usually 0.05-0.1%
depending on type of tissue and fixation.
 Incubation time could be 5-30 minutes and
10-15 minutes is commonly used.
 Incubation temperature is usually at 37 °C.

12. Heat-induced Epitope Retrieval
(HIER)
 Instruments include:
 microwave ovens
 pressure cookers
 vegetable steamers
 autoclaves
 water baths

13. Principle of HIER
 FirstTheory – Shi 1991
 Methylene bridges produced by formaldehyde are
cleaved and polypeptide chains extended to
expose their hydrophobic regions.
 During cooling process, the polypeptide chains
rapidly refold.
 Hydrophobic attractive force and electrostatic
repulsion force based on positively or negatively
charged polypeptides may balance to prevent
intertwining of polypeptide chains and expose
antigenic determinants for antigen-antibody
interaction

14. Heat-induced antigen / epitope
retrieval mechanisms

15. Principle of HIER
 2nd Theory – Morgan 1997
 Calcium coordinated complex formed during
fixation prevent antibody-antigen reaction.
 Hydroxy-methyl groups and other oxygen-rich
group (carboxyl or phosphoryl groups) interact
with calcium ion
 Produce large coordinate complex that mask
epitope.
 High temperature weakens or breaks the calcium
coordinated bonds.

16. Key Factors to be Considered
When Performing HIER
 Temperature of retrieval solution should be
around 95 °C.
 Incubation time should be at least 10 minutes
and it is usually around 20 minutes.
 pH value of retrieval solution is depending on
which solution you are using.

17. IHC images show the detection of p27 in paraffin-embedded human prostate cancer sections following
incubation of tissue for 10 minutes at 95 °C in the specified antigen retrieval solution. Compared to no HIER
treatment, p27 detection was enhanced following incubation in neutral (pH 7.0) and basic (pH 9.5) but not acidic
(pH 5.0) antigen retrieval solution. P27 was detected using anti-human/mouse/rat p27 antibody.

18. Combination of HIER and
Enzyme Method
 Alternative approach to unmask antigens if other
methods did not work
 Principle:
 Heat pretreatment increases the sensitivity of sections
to subsequent proteolytic enzyme digestion.
 Proteolytic enzyme digestion increases the sensitivity
of sections to microwaves antigen retrieval.
 Therefore, proteolytic enzyme digestion can be
carried out before or after heating.

19. Antigen Retrieval Technique
 There are several techniques available, four of
them are:
 Proteolytic enzyme digestion (PIER)
 Trypsin
 Pepsin
 protease
 Microwave enzyme digestion (HIER+Enzyme)
 Microwave and trypsin antigen retrieval
(HIER+Enzyme)
 Pressure cooker antigen retrieval (HIER)

20. Proteolytic enzyme digestion -
Trypsin
Incubate sections in pre-warmed distilled water at 37ºC
Prepare 0.1% trypsin in 0.1% calcium chloride in distilled water (37C). Adjust pH
to 7.8 using 0.1M sodium hydroxide solution.
Incubate the sections in the trypsin solution for 10 minutes at 37C
Wash section in cold running tap water to prevent further digestion
Proceed with immunostaining method of choice

21. Proteolytic enzyme digestion -
Protease
Incubate sections in pre-warmed distilled water at 37ºC
Prepare 0.1% protease in distilled water (37C). Adjust pH to 7.8 using 0.1M
sodium hydroxide solution.
Incubate the sections in the protease solution for 6 minutes at 37C
Wash section in cold running tap water to prevent further digestion
Proceed with immunostaining method of choice

22. Proteolytic enzyme digestion -
Pepsin
Incubate sections in pre-warmed distilled water at 37ºC
Prepare 0.4% pepsin in 0.01M hydrochloric acid (pH2.0) at 37C.
Incubate the sections in the trypsin solution for 15-60 minutes at 37C
Wash section in cold running tap water to prevent further digestion
Proceed with immunostaining method of choice

23. Combination of trypsin digestion and
microwave antigen retrieval
Incubate section in pre-warmed distilled water at 37C
Prepare 0.1% trypsin in 0.1 calcium chloride in distilled water at 37C. Adjust pH to 7.8 using sodium
hydroxide
Incubate the sections in the trypsin solution for 3o seconds at 37C
Wash sections in cold running tap water to prevent further digestion
Using a plastic staining rack, place the sections in 600ml of 0.01M citrate buffer pH 6.0. use a
microwaveable plastic container
Irradiate on high power (800W) for 15minutes
Carefully remove the container from the microwave and flood with cold water
Proceed with the immunostaining method of choice

24. Pressure cooker antigen retrieval
Add 1.5 liters of appropriate antigen retrieval buffer into the pressure cooker and
bring to boil (without lid)
When the antigen retrieval buffer is boiling, carefully place the slides racks into the
hot solution and seal the lid
Allow the pressure cooker to reach full pressure (15psi).Then incubate for two
minutes.Timing start only when full pressure is reached.
Transfer the pressure cooker to a sink and run cold water over the lid until all of the
pressure is released.
Flood the pressure cooker with cold water. Do not remove the slides until cool
Proceed with the immunostaining method of choice

26. General steps in immunohistochemistry

27. Staining LSAB-peroxidase
Incubate section
slide in the oven at
56C for 30min
Bring section to
water
Trypsinise the
section at 37C for
30minutes (AG
retrieval)
Undergo
proteination based
on the type of
reagent
Wash with distilled
water
Put into 3%
hydrogen peroxide
Wash with distilled
water
Rinse with buffer
solution
Incubate with
primary antibody
30min
Insert antibody link
15min
Wash with buffer
solution
Insert streptavidin
for 15min
Wash with buffer
solution
Rinse with distilled
water
Put substrate until
developing brown
colour and leave
10min
Wash with distilled
water
Stain with H&E
Rinse with water
Rinse with 70%
alcohol
DCM
Result: Grey formation

29. Labeled Streptavidin Biotin
principle
 The first layer is unlabeled primary antibody.
 The second layer is biotinylated secondary
antibody.
 The third layer is Enzyme-Streptavidin
conjugates to replace the complex of avidin-
biotin peroxidase.
 The enzyme is then visualized by application
of the substrate chromogen solutions to
produce different colorimetric end products.

31. References
 J.D Bancroft and A Steven (2008)Theory and
Practice of HistologicalTechniques (6th ED)
Churchill Livingstone.
 J.Ochei and A Kolhatkar (2000) Medical
Laboratory Science,Theory and Practice (4th
ED).TheTata McGraw Hill Company.