This document discusses immunohistochemistry, which is the localization of antigens in tissue sections using labeled antibodies. It describes the key requirements and processes for immunohistochemistry, including tissue fixation, antigen retrieval, staining procedures, and visualizing the reaction products. Potential pitfalls like false positives and negatives are also outlined. The document focuses on immunohistochemistry techniques for human tissue sections.

1. 2.3. IMMUNOHISTOCHEMISTRY
2.3.1. INTRODUCTION
Immunohistochemistry is the localization of antigens in tissue sections by the use of labeled
antibodies as specific reagents through antigen-antibody interactions that are visualized by a
marker such as fluorescent dye, enzyme, radioactive element or colloidal gold (IHC world,
2012).

2. Immunohistochemistry has become a crucial technique and widely use in many medical
research laboratories as well as clinical diagnostic. The essential conditions for
immunohistochemistry are,
 Preservation of antigen in tissues
 Antigen retrieval in fixed tissues
 Visualizing end products of reaction
 Absence of non-specific staining (IHC world, 2012).

3. 2.3.2. REQUIREMENTS FOR IMMUNOHISTOCHEMISTRY
(1) FIXATION
Tissue preparation is the cornerstone of immunohistochemistry. To ensure the preservation of tissue
architecture and cell morphology, prompt and adequate fixation is essential. However, inappropriate or
prolong fixation may significantly diminished the antibody binding capacity. The most common fixatives used
for immunohistochemistry are the followings:
(a) 4% paraformaldehyde in 0.1 M phosphate buffer
(b) 2% paraformaldehyde with 0.2% picric acid in 0.1 M phosphate buffer
(c) PLP fixative : 4% paraformaldehyde, 0.2% periodate and 1.2% lysine in 0.1M phosphate buffer
(d) 4% paraformaldehyde with 0.05 % glutaraldehyde (IHC World, 2012).

4. (2) Antigen retrieval in fixed tissue
Washing
The simplest form of reversing the effects of formalin is to wash the tissue well before processing
(IHC World, 2012).
Antigen retrieval by different methods
The demonstration of many antigens can be significantly improved by pretreatment with antigen
retrieval reagent that break the protein the protein cross-links formed by the formalin fixation and thereby
undercover hidden antigenic sites. The techniques involved the application of heat for varying lengths of
time to formalin-fixed, paraffin-embedded tissue sections in an aqueous solution. This is called heat
induced epitope retrieval (HIER). Another method uses enzyme digestion and is called proteolytic induced
epitope retrieval (PIER) (IHC World, 2012).

5. Microwave oven, pressure cooker and steamer are the most commonly use
heating methods. Other devices also include the use of autoclave and water bath.
The heating length of 20 minutes appears to be the most satisfactory and cooling
usually takes about 20 minutes. Citrate buffer pH 6.0 is the most properly used
retrieval solution and is suitable for the most of antibody applications (IHC
World, 2012).

6. Staining procedure
The following are recommendations for the improvement of staining results
and prevention of technical mishaps during the procedure (Nadji and Morales, 1990).
Attachment of sections to slides
Use of subbed or glue coated slides decrease the possibility of specimens
detachment. For freshly cut paraffin sections, overnight incubation in 37˚ C oven
minimized the loss of tissues during the procedure (Nadji and Morales, 1990).

7. Clearing and rehydration
These steps are for removal of the paraffin by a clearing agent such as
xylene and rehydration of tissue by their immersion decreasing grade of ethanol.
Shortcut in these steps will result in poor morphologic resolution of the final
products (Nadji and Morales, 1990).

8. Inhibition of endogenous peroxidase activity
The activity of endogenous peroxidase can be inhibited by several means.
The technique most commonly used is immersion of the slides in a freshly
prepared 1:4 dilution of 30 percent hydrogen peroxide in absolute methanol
(Nadji and Morales, 1990).

9. Incubation with the primary antibody
The optimal incubation time for most primary antibodies is 20 to 30
minutes at room temperature (20˚ to 22˚ C). Overnight (18 hours) incubation of
sections at 4˚ C will increase the sensitivity of the procedure with some
monoclonal antibodies, particularly those against cell membrane antigens (Nadji
and Morales, 1990).

10. The optimal concentration of a primary antibody should be determined by applying
serial dilutions of that antibody to several sections of a known positive tissue. The
slide with the best specific reaction and the least background staining reflects the
optimal dilution for that antibody. Prediluted antibodies, such as those in
commercial staining kits, are usually titrated for a tissue with an average amount of
antigen and may not be suitable for tissues containing either more or less than that
amount (Nadji and Morales, 1990).

11. Linking antibody and peroxidase conjugates
Once the optimal dilutions are determined for a batch of linking antibody and
peroxidase containing complex, such as PAP and avidin-biotin-peroxidase, they remain
constant for their use with all primary antibodies. The optimal incubation time for
linking antibodies and peroxidase conjugates is 30 to 60 minutes at room temperature
(Nadji and Morales, 1990).
Color development
Common chromogens for peroxidase are diamino benzidine (DAB) and amino
ethyl cabazol (AEC). DAB and AEC solutions should be made fresh immediately
before their use (Nadji and Morales, 1990).

12. (3) Visualizing the end products of reaction
Antigen – antibody binding is not visible under the light or electron
microscope, unless the antibody is tagged with a label that permits its
visualization. In immunohistochemistry, the labels are either directly bound to
primary, secondary or tertiary antibodies, or they are directly introduced into the
reaction by the use of other substances such as hapten, biotin or protein A (Nadji
and Morales, 1990).

13. (4) Absence of non-specific staining
Non-specific binding of immunoreagents to tissue is a universal
problem.
Causes of non-specific staining
 Charged sites: Antibodies carry a negative charge and can bind to
positively charged sites in the tissue substrate

14.  Hydrophobic reactions: Even in fixed tissue, hydrophobic bonds can be
formed with any immunoreagent, whether labeled or not.
 Fc receptors: In fresh tissue, tissue receptors for the Fc portion of antibodies
may pose an additional problem. These Fc receptors present on several cell
types such as macrophages and monocytes as part of the natural immune
defense mechanism, are largely destroyed by formalin fixation and tissue
processing. If necessary, Fab fragments of antibodies which lack the Fc portion
should be used (Nadji and Morales, 1990).

15. Prevention of non-specific binding
All the above tissue factors should be blocked by applying normal (non-
immune) serum in high concentration to the tissue preparation before applying
the specific primary antibody. Normal serum contains enough natural antibodies
and other proteins to occupy all these binding sites and prevent attachment of
the specific antibody (IHC World, 2012).

16. Polyclonal antibodies should be used as highly diluted as possible to reduce
the concentration of proteins that might bind non-specifically. Special control must
be run in order to test the protocols and for the specificity of antibody being used
(IHC World, 2012).
Positive control is to test a protocol or procedure and make sure it works. It
will be ideal to use the tissue of known positive as control. If the positive control
tissue showed negative staining, the protocol or procedure need to be checked until a
good positive staining is obtained (IHC World, 2012).

17. Negative control is to test for the specificity of antibody involved.
First, no staining must be shown when omitting primary antibody or
replacing a specific primary antibody with normal serum (must be the
same species as primary antibody). This control is easy to achieve and
can be used routinely in immunological staining (IHC World, 2012).

18. 2.3.3. ADVANTAGES OF IMMUNOHISTOCHEMISTRY
The advantages of immunohistochemistry are obvious:
1. Remarkable sensitivity and specificity.
2. Applicability to routinely processed material (even if stored for long periods).
3. Feasibility of an accurate correlation with the traditional morphologic parameters.
It is compatible with most of the fixatives currently in use and is feasible even in
decalcified material or in previously stained microscopic sections.
4. Compatible with most of the fixatives in current use.
5. Feasible with decalcified material.
6. Feasible even in previous stained microscopic sections.
7. Can be adapted to cytologic preparations and electron microscopy.
8. Can be use in conjunction with conventional techniques (such as silver stains) in
the same sections (Rosai, 2011).

19. 2.3.4. Pitfalls of immunohistochemistry (Rosai, 2011)
These can be avoided by scrupulous technique, periodic checking of the antibody
activity and proper use to positive and negative controls.
False-negative results can occur when:
1. An antibody is inappropriate, denatured, or used at the wrong concentration.
2. There is loss of antigen through autolysis and/or diffusion.
3. Presence of antigen is at a density below the level of detection with the reagents and
techniques used, because of either minimal production or excessive release.

20. False-positive results can result from:
1. Cross-reactivity of the antibody with antigens different from the one being
sought.
2. Nonspecific binding of the antibody to the tissue.
3. Presence of endogenous peroxidase in cell.
4. Entrapment of normal tissues by the tumor cells.
5. Release of soluble proteins from the cytoplasm of normal cells invaded by the
tumor, with nonspecific absorption by the tumor cells.

21. 2.3.5. Detection system for human tissue sections N-histofine simple stain
MAX PO
Feature
N-histofine Simple Stain MAX PO is a detection reagent designed
specifically to allow immunohistochemical staining on formalin-fixed
paraffin-embedded human tissue sections. It is the labeled polymer prepared
by combining amino acid polymers with peroxidase (PO) and secondary
antibody which is reduced to Fab fragment. To eliminate background
staining, solid-phase absorption of secondary antibody is carried out with
human serum.

22. Advantages
1. Simplified staining steps
2. High sensitivity
3. Unaffected by endogenous biotin in the tissue
4. Ready to use