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1. Immunohistochemistry:
techniques and considerations

2. Immunohistochemistry
• What is it?
• What can it tell you?
• What are its limitations?
• How is it done?
• What are the controls?
• Quantification
• Single vs double or triple labelling

3. Antibodies and Antigens
www.IHCworld.com
Fab region
Paratope + Epitope
Ab-Ag complex

4. What is immunohistochemistry?
– Immunohistochemistry is the localization in tissues
of an (known) antigen by means of antibodies
directed towards that (specific) antigen.
– Ab-Ag complex is visualised using a marker.
– Immunocytochemistry refers to localization in isolated cells or labelling
directed to cell specific compartment (e.g. the cell membrane, Golgi or
lysosomes, etc.).

5. Immunohistochemistry
• What can it tell you?
– Tissue or cellular localization of an antigen with
cellular or subcellular resolution.
– Specific cell or tissue expression of an antigen.
– Major (but probably not minor) changes in the
expression pattern of an antigen.
– Dynamic changes in the expression of an antigen;
particularly useful in animal models of disease

6. Immunohistochemistry
• What are its inherent limitations?
– Virtually nothing about quantity of antigen.
– Despite doing rigorous controls, it cannot really tell
you that an antigen is in the tissue. It can only
show “immunoreactivity” or “like-
immunoreactivity”.
This distinction is often overlooked.

7. Immunohistochemistry
http://en.wikipedia.org/wiki/Immunohistochemistry
• The theory

8. How is IHC done?
• Preparation of Ab’s
• Preparation of tissue
– Fixation
– Presentation
– Chemical preparation
• 1° Ab application
• Visualisation of 1° Ab

9. Preparation of antibodies
• Largely commercial
• Often of spurious quality and variable titres
• Rarely well-characterized
– Let the literature be a guide, but do not hesitate to telephone or
email potential sources or people in the field to get advice.
• Polyclonal
– Ag injected into host animal. Serum collected and purified.
– Multiple antibodies produced by different cell types bind multiple
epitopes on Ag
• Monoclonal
– Ag injected into mouse. Lymphocytes isolated, hybridized.
– One antibody produced by one cell type binds one epitope on Ag.

10. Preparation of tissue - fixation
Prevention of Ag translocation and/or degradation
• Lots fixatives and they ALL potentially alter the presentation
of antigen, because they crosslink protein and/or
carbohydrates in the cell.
• Most common fixatives are aldehydes (formalin,
paraformaldehyde, glutaraldheyde (EM), Zamboni’s fixative).
• Alcohols and acetone can be used, but they precipitate
proteins and so tend to be good for cell-surface antigens.
• Fixation can be performed at the level of the animal, tissue
samples, e.g. biopsies, or after sectioning or mounting of fresh
tissues or cells on a slide or in a dish.

11. Preparation of tissue - fixation
• Whole animal
– Perfuse via vasculature – typically intracardiac.
– ADVANTAGES:
– Removes blood
– Ag degradation prevented before dissection – improves
specimen quality
– Effective fixation
– All tissues fixed
– DISADVANTAGES:
– Time consuming
– Messy
– Safety concerns – use fume hood
– Uses a lot of fixative

12. Preparation of tissue - fixation
• Tissue specimens
– Immersion fixation.
– ADVANTAGES:
– Quick, easy and safe
– Excellent for small samples
– Can prepare and manipulate tissue prior to fixation eg
flat mounts of gut tissue
– DISADVANTAGES:
– Blood present
– Large samples may take long time – not effectively
fixed

13. Preparation of tissue - fixation
• Cell preparations, fresh-frozen tissues mounted onto slides
– Immersion fixation.
– ADVANTAGES:
– Quick, easy and effective
– Fast for diagnostics
– Good for cell surface markers
– Rapid fixation
– DISADVANTAGES:
– Fresh frozen tissues need to be handled by skilled
technician as morphology can suffer and degradation
can occur

14. Preparation of tissue - embedding
• Embedding - Paraffin or Cryostat
– Preserve tissue integrity when sectioning
– Paraffin infiltrates tissues
– Cryo-embedding requires cryoprotection
– Sucrose
– Prevents ice crystals forming before embedding and
freezing

15. Preparation of tissue - sectioning
• Thickness main consideration – confocal microscopy
revolutionised visualisation in thicker tissues
• Sectioning:
• Sledge microtome – paraffin
• Cryostat – frozen
• Vibratome – thicker tissues don’t require embedding
• Whole mount:
• Flat mount – no sectioning
• Useful for visulaisation laminar structures eg enteric
nervous system

16. Preparation of tissue
• Improving antibody penetration
– Reduce crosslinking
– Reduce membrane barriers
– Improve the presentation of the antigen
• Antigen retrieval for paraffin embedding
– Heat and/or proteolysis
• DMSO/Ethanol washes
• Triton X or other detergents to reduce surface
tension therefore use less reagent

17. Blocking steps
• Blocking unwanted tissue antigens
– Improves signal to noise by preventing Fc receptor
binding
– Use normal serum of host species of the secondary
antibody prior to 1° Ab incubation to reduce
hydrophobic interaction of cross-linked proteins
and Ab
• Blocking unwanted endogenous peroxidase
– Visualise with DAB substrate
– Eliminate with hydrogen peroxide

18. Antibody application
• Incubation with primary antibody
– Floating immersion
– On slides on a stage in humid chamber
• Conditions should include consideration that
they degrade
• Use the lowest effective dilution, which needs
to be determined empirically

19. Direct immunofluorescence
• One step
• Ab is labeled with fluorescent tag
• Technique is fast
• Low sensitivity due to lack of signal amplification

20. Indirect immunofluorescence
• Two steps
• Step 1 – bind 1° Ab to Ag
• Step 2 – bind fluorescent labeled 2° Ab to 1° Ab
• Sensitive – signal amplification
• Use one 2° Ab for many unlabeled 1° Ab

21. Indirect immunoenzyme
• Peroxidase anti-peroxidase or
PAP method
• Three steps
• Step 1 – bind 1° Ab to Ag
• Step 2 – bind unconjugated 2° Ab
to 1° Ab
• Step 3 – bind PAP complex to 2°
Ab
• Sensitive – signal amplification
• Use DAB as a substrate for
peroxidase  colourimetric end
product
http://www.biologie.uni-regensburg.de/Zoologie/Schneuwly/Hofbauer/DROSI/strentw42.htm

22. Indirect immunoenzyme
• Avidin-Biotin-Complex or ABC
method
• Three steps
• Step 1 – bind 1° Ab to Ag
• Step 2 – bind biotinylated 2° Ab
to 1° Ab
• Step 3 – bind avidin-biotin-
peroxidase complex to 2° Ab
• Very sensitive – signal
amplification
• Use DAB as a substrate for
peroxidase  colourimetric end
product
http://www.biologie.uni-regensburg.de/Zoologie/Schneuwly/Hofbauer/DROSI/strentw42.htm

23. Capturing images
• Take convincing images!!
• If the picture isn’t good you may as well
have not done the experiment.
• Consider composition and make sure you
take representative images.
• DO NOT MANIPULATE IMAGES

24. PGP9.5 immunoreactivity in rat nodose ganglia

25. Controls
• Method specificity
– Controls for tissue, reagents, secondary antibodies,
autofluorescence, non-specific fluorescence,
endogenous enzymes, etc., etc.
• APPROACH - Leave out the primary antibody, do
everything else the same.
• Negative control – no staining – if you see something
there is a problem.
• IT IS NOT A CONTROL FOR THE SPECIFICITY OF THE
LABELLING THAT YOU DO SEE!!

26. Controls
• Antibody specificity
– Controls for the specificity of the reaction
• APPROACHES
1. Preabsorb the primary antibody with the immunizing antigen
(question a company which does not supply this)
2. Replace the primary with preimmune serum from the same
animal that the primary was raised in – or control ascites fluid
for monoclonals
• Negative control – no staining – if you see something
there is a problem.
• IT IS A CONTROL FOR THE SPECIFICITY OF THE LABELLING
THAT YOU SEE – BUT – IT DOES NOT SAY THIS IS THE
REAL ANTIGEN.

27. PPARa immunoreactivity in
rat ileum
PPARa antibody
preabsorbed in blocking
peptide in rat ileum

28. Controls
• Antibody specificity
– Positive controls
• APPROACHES
1. Label tissues and/or transfected cells that are known to
express the antigen of interest
• Can be very good for well-established antigens, but is
limited for novel antigens
• Strongly encouraged as it is “positive”

29. Controls
• Antibody specificity
– KO MICE – a new approach that is gaining
popularity
• APPROACHES
1. Label tissues in mice with and without the gene product of
interest
2. No labelling in the KO mouse is very powerful and speaks
highly to the specificity of the reagents
• Great if you can do it.
• Note that not all gene products are actually knocked out
fully.

30. Quantification in immunohistochemistry
• Know the limits of the technique!
• Can readily define labelled area
• Can readily define the number of labelled cells if
you can count total cells or some subset of cells
• Much harder to define amount of labelling and
this should be avoided unless strictly necessary
and then only when carefully performed. Note
that linearity of a signal is hard to prove.

31. GFAP expression is enhanced in colitis
in GFP-GFAP transgenic mice

32. Single vs Double or Triple labelling
• Sophisticated and fraught with difficulties that are
not always appreciated.
– How to incubate primary antibodies
• Sequential vs combined
– Interactions with secondary antibodies
• Additional controls are required
– “Bleed through” of fluorochromes
• Need to assess filters
• Consider sequential vs concurrent scanning when using a
confocal microscope
• If one reaction is very intense it can “bleach” a much weaker
one in an overlay

33. Single vs Double or Triple labelling
– Consider the species and choice of reagents
carefully.
• Must test specificity of secondary antibodies – cannot assume they
not cross-react. Rigorous testing is recommended.
– Primary A vs Secondary A
– Primary B vs Secondary A
– Primary B vs Secondary B
– Primary A vs Secondary B
– Primary A + Primary B vs Secondary A
– Primary A + Primary B vs Secondary B
– Primary A + Primary B vs Secondary A + Secondary B
– Primary A + Secondary A + Secondary B
– Primary B + Secondary A + Secondary B

34. PPARa immunoreactivity is
localised to the nuclei of
neurons in rat ileum

35. Neurobiotin Calbindin Merged image
50µm
Intrinsic primary afferent neurons of
the myenteric plexus are Dogiel Type
II neurons that express calbindin

36. IL-1β activates neurons and glia in
the submucosal plexus of the ileum.

37. Red (CY3): rat anti-substance P
Green (FITC): mouse anti-VIP
Blue (AMCA): rabbit anti-CGRP
Distribution of nerves in the guinea pig ileum submucosal plexus
TRIPLE LABELLING

38. Blue - DAPI; Green (FITC): rabbit anti-CART; Red (CY3): goat anti-leptin; Purple (false
coloured, CY5): rat anti-NPY
Rat brain section – confocal microscopy QUADRUPLE Labelling

39. Some useful websites
• http://www.uchsc.edu/rmtsc/protocol/index.html
• http://www.jacksonimmuno.com/
• http://www.invitrogen.com/site/us/en/home/brands/Molecular-Probes.html
• http://www.biocompare.com/ProductCategories/2045/Antibodies.html
• http://www.cytochemistry.net/Cytochem.htm
• http://www.sigmaaldrich.com/life-science/cell-biology/antibodies.html