Antigen Retrieval Methods &
Controls in IHC
Dr. Usman Shams
• Formaldehyde forms methylene bridges
between proteins, which can hinder epitope
recognition by the primary antibodies.
•...
HIER
• HIER is the most common approach to antigen retrieval.
• Temperature, pH and time of incubation are critical
factor...
• Microwave oven are mostly used for this purpose.
• The time length of 15-20 minutes appear to be most
satisfactory and a...
PIER
• The PIER approach utilizes the enzymatic activity of
pronase, pepsin, ficin, trypsin or proteinase K to partially
d...
• Antigen retrieval can be performed using either HIER or
PIER, or through a combination of both approaches.
• Frozen tiss...
• Williams and coworkers137 investigated the effect of
tissue preparation on IHC staining by using tonsil tissues
that wer...
• Fraenkel-Conrat and coworkers, who documented that
the chemical reactions that occur between protein and
formalin may be...
Immunohistochemistry Methods
CONTROLS:
 Special controls must be run in order to test the
protocols and foe the specifici...
CONTROLS
1. POSITIVE CONTROLS
 It is taken from the known positive case.
 If the positive control tissue showed negative...
CONTROLS
2. NEGATIVE CONROLS
 Negative control is test for the specificity of the antibody.
 First the negative staining...
Antigen retrieval
Upcoming SlideShare
Loading in …5
×

Antigen retrieval

883 views

Published on

Published in: Education
  • Be the first to comment

  • Be the first to like this

Antigen retrieval

  1. 1. Antigen Retrieval Methods & Controls in IHC Dr. Usman Shams
  2. 2. • 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).
  3. 3. HIER • 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 steamer).
  4. 4. • 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.
  5. 5. PIER • 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.
  6. 6. • 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.
  7. 7. • 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.
  8. 8. • 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).
  9. 9. Immunohistochemistry Methods CONTROLS:  Special controls must be run in order to test the protocols and foe the specificity of the antibody being used.  Two types of controls are used 1. Positive controls 2. Negative controls
  10. 10. 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.
  11. 11. CONTROLS 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.

×