This document provides an overview of immunohistochemistry (IHC), including its history, basic methods, considerations, and applications. IHC combines histology, immunology, and biochemical techniques to visualize the distribution and localization of specific cellular components within tissues through antigen-antibody reactions tagged with visible labels. Key steps discussed include fixation, sectioning, antigen retrieval, blocking, controls, direct and indirect antibody binding methods, detection methods including enzymatic and fluorescence techniques, and important considerations for optimizing IHC experiments.

1. BASICS OF
IMMUNOHISTOCHEMIST
RY
Submtted By
Dr. Aderao Ganesh N.
P-2063
Animal Nutrition Division
Submitted to
Dr. Vikash Chandra
Sr. Scientist
Department of Physiology and Climatology

2. COMMON METHODS OF PROTEIN
DETECTION
 ELISA
 Gel Electrophoresis
 Western blot
 Immunoprecipitation
 Spectrophotometry
 Enzyme assays
 X-ray crystallography
 NMR
 Immunohistochemistry

3. WHY
IMMUNOHISTOCHEMISTRY..??
 Antibodies bind to antigen in specific manner
 Gives information about spatial location
 Can be used to locate particular cells and
proteins
 Can be used to identify cellular events – e.g.
apoptosis

4. WHAT IS
IMMUNOHISTOCHEMISTRY..??
 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.

5. HISTORY
 The principle has existed since the 1930s.
 Started in 1941 when Coons identified pneumococci using
a direct fluorescent method.
 Indirect method
 Addition of horseradish peroxidase
 Peroxidase anti-peroxidase technique in 1979
 Use of Avidin & Biotin complex in early 1980’s

6. CELLULAR ANTIGENS CAN
WE TARGET?
 Cytoplasmic
 Nuclear
 Cell membrane
 Lipids
 Proteins

7. IMPORTANT CONSIDERATIONS FOR
IHC
 Antibody selection
 Fixation
 Sectioning
 Antigen Retrieval
 Blocking
 Controls
 Direct method
 Indirect method
 Immunoenzyme
 Fluorescence
 Multiple labeling
You actually need to care about all this now because it may affect
how you harvest your samples !

8. MONOCLONAL V. POLYCLONAL
 Monoclonal
 Mouse or rabbit
hybridoma
 Tends to be ‘cleaner’
 Very consistent batch-
to-batch
 More likely to get false
negative results
 Polyclonal
 Many different species
 Tends to have more
non-specific reactivity
 Can have very
different
avidity/affinity batch-
to-batch
 More likely to have
success in an
unknown application

9. STEPS OF
IMMUNOHISTOCHEMISTRY

10. FIXATION
 To preserve tissue morphology and retain the
antigenicity
 of the target molecules
 To avoid proteolytic changes
 Helps to prevent
 Elution
 Degradation •
 Modification
 Preserves the position of the Ag
 Preserves the secondary and tertiary structure to a
possible extent
 Provides target for Ab molecules
 Formaldehyde is the preferred fixative as it keeps all
the proteins intact and is cheaper one

11. FIXATION
 Aldehyde
 10% NBF
 4% formaldehyde with
PBS buffer
 2% formaldehyde with
picric acid and PBS
 The paraformaldehyde
paradox
 Immersion v.
transcardial perfusion
 24-72 hours
 Best for good
architecture
 Frozen
 LN2
 With or without sucrose
 Fix with Alcohol,
acetone or methanol
(fix by coagulation, also
permeabilizes)
 Best for cell membrane
antigens, cytokines

12. CHEMICAL FIXATIVES
 Simple fixatives
 Formalin
 Mercuric chloride
 Osmic acid
 Picric acid
 Acetone
 Ethyl alcohol
 Osmium tetaoxide
 Campound fixatives
Microanatomical
Formal saline
Neutral buffere
formal saline
Zenkers fluid
Bounes fluid
Cytological
Nuclear
Carnoys fluid
Cytoplasmic-
Chamoys fluid

13. SECTIONING
 Paraffin
 Must heat and process
through xylenes and
alcohols – ruins some
antigens
 Most commonly used
 BEST if not stored
more than two weeks
– lose antigenicity
after that time
 Frozen
 Better survival of
many antigens
 Poor morphology
 Poor resolution at
higher mag
 Special storage
 Cutting difficulty
Done to have better picture and thin layer of cells for better
processing and better result

14. ANTIGEN RETRIVAL
 To prepare the tissue to receive the primary antibody
 Ables the partial reversal of formaldehyde induced
confirmational change of Ags
 Increases the accessibility of the Ab to the Ag
 Two methods:
 Heat
 Enzyme digestion
 Choice of Ag retrieval depends on the Ag to be
demonstrated
 Heat Induced Epitope Retrieval (HIER) is widely
used.

15. ANTIGEN RETRIEVAL
 HIER (Heat induced
epitope retrieval )
 Use
MW/steamer/pressure
cooker ~ 20 minutes,
slow cool
 Citrate 6.0
 Tris-EDTA 9.0
 EDTA 8.0
 Must determine for
each new
antibody/antigen
target
 PIER (Protease
induced epitope
retrieval )
 Proteinase K
 Trypsin
 Pepsin
 Pronase,etc.
 Destroys some
epitopes
 Bad for morphology

16. IMPROVING ANTIBODY
PENETRATION
 Need this for intracellular (cytoplasmic, nuclear) or
membrane components when epitope is inside cell
membrane
 Detergents most popular
 Triton-X
 Tween
 Also decreases surface tension – better coverage
 Can’t be used for membrane proteins (surface tension
reduced)
 Acetone/Methanol
 Precipitate proteins outside cell membranes- more
accessible
 Saponin
 Punches holes in cell membrane – holes close up when
removed

17. BLOCKING
 Blocking is done to avoid binding of primary
antibodies to non specific sites and hamper result.
 Done by BSA or 10% serum
 Background staining
 Specific
 Polyclonal antibodies – impure antigen used
 Inadequate fixation – diffusion of antigen – often
worse in center of large block
 Non-specific
 Non-immunologic binding – usually uniform
 Endogenous peroxidases
 Endogenous biotin

18. NON-SPECIFIC STAINING
Before blocking After blocking

19. CONTROLS
 Controls should be kept in order to have better
estimate and specificity.
 Positive control
 Best is tissue with known specificity
 Negative control
 Best is IgG from same species immunized against
non-biologic molecule – e.g. BRDU when no BRDU is
present in tissue
 Can also use non-immunized serum from same
species

20. ANTIBODY BINDING
 Direct Method
Labelled Ab reacts directly with Ag in tissue
sections
Single step method
Short and quick
Insensitive due to little signal amplification
E.g., FITC conjugated Antisera
Herein the antibodies are incubated for 30-60
min.

21. DIRECT METHOD-
PRIMARY ANTIBODY ONLY

22. ANTIBODY BINDING - INDIRECT
METHOD
 Unlabelled Primary Ab reacts with Ag and the
labelled secondary Ab reacts with the primary Ab
 Sensitive due to signal amplification
 Economical as single secondary Ab can be used
against many Abs from same species
 Peroxidase Anti-Peroxidase/ Alkaline
Phosphatase Anti-Alkaline Phosphatase (PAP/
APAAP) Method
 Avidin-Biotin Complex (ABC) Method
 Streptavidin – Peroxidase Method

23. INDIRECT METHOD – PRIMARY AND
SECONDARY ANTIBODIES
Goat anti-actin
Donkey anti-goat
labeled with 488

24. ENZYME LINKAGE INDIRECT
METHOD

25. MULTIPLE
IMMUNOFLUORESCENCE

26. DETECTION METHODS
 Ag-Ab conjugates are visualized by the use of a
label
 Enzymes that produce a colored precipitate in
the presence of a substrate are used as labels
 Labels :
Peroxidase
 Alkaline Phosphatase
 Detection systems:
Direct or Single step Method
Indirect or Two step Method

27. MULTIPLE LABELLING OF A TISSUE SECTION

28. ENZYMATIC DETECTION
METHODS
 Brightfield microscope sufficient for analysis of
specimens
 Suitable for tissue analysis at low magnification
 Resolution of subcellular structures not as good as with
fluorescence methods, but can be combined with electron
microscopy
 Unimited shelf life of labelled specimens
 Substrate reagents often toxic/carcinogenic

29. PAP METHOD
(PEROXIDASE ANTI-PEROXIDASE
METHOD)

30. ABC METHOD

31. SUMMARY
 IHC = immunology +histology + chemistry
 Has strengths and weaknesses
 Think about your planned assay before acquiring
tissue
 Good block, appropriately fixed and sectioned can
give you great data
 Bad block, inappropriately fixed and sectioned,
can give you misleading data and waste money

32. SUMMARY
 Very efficient and sensitive method for localizing
of antigens and their identification
 Although, advances has been occurred for protein
identification but this is used till now due to its
benifit that it identifies the protein in intact cell.
 Use of secondary antibody is always good as it
will be less expensive and yield good results.
 IHC should can be used in the cells in their
natural state.