Immunohistochemistry (IHC) utilizes labeled antibodies to localize specific antigens in tissue sections through antigen-antibody interactions visualized by markers like fluorescent dyes or enzymes. IHC allows visualization of the distribution and localization of cellular components within tissues. The process involves raising antibodies to target antigens, labeling the antibodies, and applying them to tissue sections using techniques like direct, indirect, or peroxidase anti-peroxidase (PAP) methods. IHC is a sensitive and specific technique that is useful for cancer diagnosis and research applications.

1. Basics of
Immunohistochemistry
Dr. Akshay Agarwal
Moderator: Dr. Shilpi Sahu
22/4/16

2. Introduction
• Immunohistochemistry (IHC) combines histological,
immunological and biochemical techniques for the
identification of specific tissue components by means of
a specific antigen/antibody reaction tagged with a visible
label.
• IHC makes it possible to visualize the distribution and
localization of specific cellular components within a cell
or tissue.

3. • IHC is an application of antibodies to tissue
preparation for the localization of target antigens:
• Wide range of specific antibodies
• Highly sensitive detection system

4. Immunohistochemistry utilizes labeled antibodies to
localize specific cell and tissue antigens, and is among the
most sensitive and specific histochemical techniques.
Because many targeted antigens are proteins whose
structure might be altered by fixation and clearing, so
frozen sections are commonly used.
However in most cases, paraffin wax can be used for

5. Immunocytochemistry
Cells grown, spun into a pellet, frozen or
paraffin embedded and sectioned
Cells grown as a monolayer
OR use tissue sections that are frozen or paraffin embedded
Sections from tissues contain many different kinds of cells
as well as extra-cellular matrix components
cells on slides
Immunohistochemistry

6. If the tissue is frozen
Tissue section on glass slide: Frozen
Acetone fixed:
- precipitates proteins onto cell surface---may extract lipids
- is needed for many of the “CD” antibodies
Unfixed:
Advantage: antigens are unaltered
Disadvantage: sections may fall off slide during staining
Paraformaldehyde fixed:
- needs to be freshly made, or frozen soon after

7. Principle
• The principle of immunohistochemistry is to localize
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.

8. Antibodies (Immunoglobulins)
• Glycoprotein that are produced by plasma
cells and used by the immune system to
identify and neutralise foreign objects, ie.
bacteria and viruses
• Recognise a specific Antigen- mainly
proteins, glycoprotein, polysaccharides
• Complementary Determining Region

9. Antigen Detection

10. Antibodies binding to Antigens

11. Antigens

12. A. Raising Antibodies:
• Repeated injection of antigens (proteins, glycoproteins,
proteoglycans, and some polysaccharides) causes the
injected animal's B lymphocytes to differentiate into
plasma cells and produce antibodies.
• Members of a lymphocyte clone (descendents of a single
lymphocyte) produce a single type of antibody, which
binds to a specific antigenic site, or epitope.

13. 1. Polyclonal antibodies: Large complex antigens may
have multiple epitopes and elicit several antibody types.
Mixtures of different antibodies to a single antigen are
called polyclonal antibodies.
2. Monoclonal antibodies: Antibodies specific for a single
epitope and produced by a single clone are called
monoclonal antibodies and are commonly raised in mice.

15. B. Labeling Antibodies:
• Antibodies are not visible with standard microscopy and
must be labeled in a manner that does not interfere with
their binding specificity.
• Common labels include fluorochromes (eg, fluorescein,
rhodamine), enzymes demonstrable via enzyme
histochemical techniques (eg, peroxidase, alkaline
phosphatase), and electron-scattering compounds for use
in electron microscopy (eg, ferritin, colloidal gold).

16. Method
• Direct Method
• Indirect Method
• PAP Method

17. Direct Method
Tissue Antigen
Labeled Antibody

18. Two-Step Indirect Method
Tissue Antigen
Primary Antibody
Secondary Antibody

19. Goat anti-actin labeled with
594

20. Goat anti-actin
Donkey anti-goat
labeled with 488

21. Enzyme linkage indirect method
Goat anti-actin
Flourochrome (488)
conjugated
streptavidin
Biotinylated
donkey anti-
goat

22. PAP Method
(peroxidase anti-peroxidase method)

23. Peroxidase Antiperoxidase Method
• Further development of indirect technique
• It involves third layer of PAP complex
• This complex is made up of 2 Ab molecules & 3 horseradish
peroxidase molecules
• Advantages –
The sensitivity is about 100 to 1000 times higher since
peroxidase molecule is not chemically conjugated to anti IgG
but immunologically bound & loses none of its enzymatic
activity
Allows use of much higher dilution of primary antibodies
• Disadvantages –
Antibody incorporated into PAP reagent should be of same
species as the primary antibody

24. Other Methods
• Alkaline Phosphatase Anti Alkaline
Phosphatase
• Avidin Biotin
• Labelled strept Avidin Biotin
• Envision System
• Fluorescyl-Tyramide Amplification

25. • Labels include :
Florescent dyes
Enzymes – Horseradish peroxidase
Colloidal gold
Radioactive element

26. Applications
• Cancer diagnostics
• differential diagnosis
• Treatment of cancer
• Research

27. General Immunohistochemistry
Protocol

28. Part 1
1. Fixation
Fresh unfixed, fixed, or formalin fixation and
paraffin embedding
2. Sectioning
3. Whole Mount Preparation
Tissue preparation

29. Part 2
1. Antigen retrieval
Proteolytic enzyme method and Heat-induced method
2. Inhibition of endogenous tissue components
3% H2O2, 0.01% avidin
3. Blocking of nonspecific sites
10% normal serum
Pre-treatment

30. Part 3
• Make a selection based on the type of
specimen, the primary antibody, the degree
of sensitivity and the processing time required.
staining

31. video

32. EXACT PROCEDURE
Deparaffinise and rehydrate
Ag retrieval
Rinse with buffer water
Block endogenous peroxidase with 3%H2O2-methanol(30min)
Antigen retreival
Incubate with primary antibody (rabbit antiserum)(30min)

33. Modified buffer(10min)
Incubate with secondary antibody (30min)
Modified buffer(10min)
Incubation with PAP complex(10min)
Modified buffer (10min)
Detection of immune reaction with DAB chromogen(5min)
Rinse with water
Counterstain with Hematoxylin(5min) and Mount

34. ADVANTAGES
• Remarkable sensitivity & specificity
• Can be done on routinely processed tissue,on
decalcified material, in previously stained
microscopic sections & even on totally necrotic
material
• Allows microbiological & morphological correlation
• Provides diagnosis when fresh tissue is not available

35. LIMITATIONS
• There is no uniformly superior method & different
modifications may be required under different circumstances
• There is paucity of antibodies with high degree of specificity
for cellular & tissue antigens
• Although there are Antigen Retrieval methods, loss of few
antigens continue.

36. Facts About IHC
• Diagnosis should be based on clinical history, radiological findings,
H & E morphology with confirmation by IHC testing
• Use of panel of IHC stains rather than over-reliance on a single
antibody is important principle
• Detection of infectious agent/identification of physiologic
substance in aberrant location directly determines diagnosis
• Negative immunoreaction in IHC never rules out diagnosis
• Key is –Utilize IHC as a cost effective tool in patient care

37. RECENT ADVANCES
• Genogenic Immunohistochemistry
• Search for proteins for targeted therapy
• Newer technology for development of more specific
antibodies
• Automation
• Tissue microarray for high throughput technique
• Shorter turn around time

38. SUMMARY
• IHC IS ESSENTIAL EXTENSION OF SURGICAL PATHOLOGY
• Even in resource poor setting it should be used for work up of
lymphoma, ER,PR & HER-2 status, diagnosis of malignant round cell
tumors, metastasis from unknown site
• Good tentative diagnosis is pre-requisite for successful IHC
• IHC IS NOT A SURROGATE FOR ROUTINE HP DIAGNOSTIC SKILL
• Developing diagnostic algorithms based on best evidence will help in
deciding the number of immunostains.

39. TUMORS 1st TIER MARKERS 2nd
TIER MARKERS
Epithelial
tumors(carcinomas)
Pankeratin HMW keratin,LMW
keratin,EMA,B72.3,CEA
Mesenchymal
tumors(sarcomas)
Vimentin Desmin,factor VIII
SPECIAL GROUP
Melanomas Vimentin & S100 HMB45,NSE
Germ cell tumor Pankeratin &vimentin AFP,HCG,Ferritin
Neural/Neuroendocr-
ine tumor
NSE & NF Chromogranin,Leu7,Synaptophy--
sin.GFAP
Lymphomas LCA Pan-B,Pan-T,kappa & lambda light
chain,R-S cell marker,histiocytic cell
marker
Antigens of value for analysis of anaplastic tumors

40. Thank You