This document summarizes immunohistochemistry (IHC) techniques. IHC combines immunological and histological methods to identify specific proteins in tissues using antigen-antibody reactions. The document discusses common IHC methods like direct, indirect, and enzyme-linked methods. It also covers important considerations for IHC like antibody selection, tissue fixation and processing, antigen retrieval, blocking, and controls. The goal of IHC is to visualize the distribution and localization of cellular components, but the quality of results depends highly on proper sample preparation.
2. Common Methods of protein
detection
ELISA
Gel Electrophoresis
Western blot
Immunoprecipitation
Spectrophotometry
Enzyme assays
X-ray crystallography
NMR
Immunohistochemistry
3. Immunohistochemistry – what’s
good about it?
Antibodies bind to antigen in specific manner
Gives you a spatial location
Can be used to locate particular cells and
proteins
Can be used to identify cellular events –
e.g.apoptosis
4. 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.
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
13. 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 !
14. Options for antibodies that
will affect your results
Monoclonal v. Polyclonal
Raised against whole molecule, N-terminus,
C-terminus, specific amino acids
Ascites, supernatant, serum
16. 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
17. Make sure your antibody is
validated for your
application!!!
IF v. IHC with fluorescence
WB, ELISA, IP, etc.
18. Whole molecule or specific
portion of epitope?
Very dependent on individual assay
20. 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
Many others
Best for good architecture
Frozen
LN2
With or without sucrose
OCT
Fix with acetone or
methanol (fix by
coagulation, also
permeabilizes)
Best for cell membrane
antigens, cytokines
22. 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
23. Antigen retrieval
HIER
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
Proteinase K
Trypsin
Pepsin
Pronase,etc.
Destroys some epitopes
Bad for morphology
24. 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 use for membrane proteins
Acetone/Methanol
Precipitate proteins outside cell membranes- more accessible
Saponin
Punches holes in cell membrane – holes close up when removed
25. Blocking
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
27. Controls
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
33. Enzymatic detection methods
Brightfield microscope sufficient for analysisBrightfield microscope sufficient for analysis
of specimensof specimens
Suitable for tissue analysis at lowSuitable for tissue analysis at low
magnificationmagnification
Resolution of subcellular structures not asResolution of subcellular structures not as
good as with fluorescence methods, but cangood as with fluorescence methods, but can
be combined with electron microscopybe combined with electron microscopy
Unimited shelf life of labelled specimensUnimited shelf life of labelled specimens
Substrate reagents often toxic/carcinogenicSubstrate reagents often toxic/carcinogenic
38. 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
Hinweis der Redaktion
The two variable regions recognise bind to antigen (parts of invading bacteria) leading to the invading bacteria being destroyed. Antibodies produced in the body are polyclonal, because each one has a different variable region and can target a different antigen (until a threat is realised in which case they massively overproduce the relavent antibody).The two variable regions recognise bind to antigen (parts of invading bacteria) leading to the invading bacteria being destroyed. Antibodies produced in the body are polyclonal, because each one has a different variable region and can target a different antigen (until a threat is realised in which case they massively overproduce the relavent antibody).
Formaldehyde fixes by formation of hydroxymethyl adducts on reactive side chains of proteins. Once sufficient adducts are formed, they slowly cross-link to stabilize the proteins in a gel-like formation. At room temperature, it takes approximately 24 hours for maximal binding of formaldehyde to occur and hence all the adducts to form. These initial adducts, and any initial cross-links formed, are unstable and easily reversed. For tissues fixed for 24 hours, the cross-links are largely reversed by washing ( in water or 70% EtOH) after a few hours. It probably takes at least 5-7 days for most of the cross-links to form, and a small amount of cross-linking still continues over time. Even after fixation for 7 days or more, the cross-links can still be reversed by prolonged washing. That's great for IHC, since cross-links can be reversed by HIER (which is just washing at elevated temperatures!).
Influence of fixation time, fixation temperature, and trypsin treatment on uPA immunoperoxidase staining of paraffin-embedded specimens of human ductal breast cancer tissue. Specimens were formalin fixed at 4° C for 1 hour (Panels a, f, g), 8 hours (Panel b), and 24 hours (Panel c) or at room temperature for 8 hours (Panel d) and 24 hours (Panel e), and immunostained with polyclonal antibodies to uPA (pAb2, 5 g/ml). Most sections were briefly predigested with trypsin (Panels a–f), although this treatment was omitted for one of the sections (Panel g). Note that when fixation was performed at 4° C, the immunostaining produced is strong in specimens fixed for 1 hour and 8 hours (Panels a, b, f), but barely detectable after fixation for 24 hours (Panel c). At room temperature fixation for 8 hours results in weak staining (Panel d), and fixation for 24 hours results in no immunostaining (Panel e). When trypsin predigestion is omitted, there is little or no uPA immunostaining, even of sections formalin fixed for 1 hour at 4° C (Panel g). Note that the immunostaining in all sections appears in the stroma and not in the cancer cells and that the strongest immunostaining is present in the fibroblast-like cells surrounding the tumor cell islands (arrows in Panel a). Bars in Panels a–e: 30 m; Panels f–g, 50 m