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Chapter 6
Detection Methods
Kenneth Petersen, PhD, MSc
Hans Christian Pedersen, MSc
Detect (n.)
To discover or determine the existence or presence of <something>.
Merriam-Webster Online Dictionary
Polymer
(Doublestain)
4
Number of Steps
iCSA
LSAB/ABC
Indirect
Polymer
+ Linker
Polymer
Direct
CSA II
Sensitivity
to HRP was used to visualize antigens in tissue using the indirect method, where a second antibody is used to recognize the
din (from chicken egg) both have four binding sites for bio-
gether via the enzyme (8). The only requirement is that the en-
79
Secondary
antibody
Primary
antibody
Tissue
antigen
Direct staining
Indirect staining
lectin-like and negatively charged tissue components at physiological pH. For streptavidin less non-specific tissue binding is
observed. Another challenge is the presence of endogenous biotin in tissues. Formalin fixation and paraffin embedding has been
Biotinylated
secondary antibody
Primary antibody
Tissue antigen
Biotinylated
secondary antibody, mouse/rabbit
Primary antibody
Tissue antigen
can be conjugated to a single dextran backbone. This construct allowed the entire IHC staining procedure, from primary antibody to enzyme, to be accomplished in a single step
(10). On the other hand, one limitation of this method was its
restriction to a select group of primary antibodies provided
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primary antibodies.
of the appropriate substrate (14). Another possibility is repetition of the biotinyl-tyramide reaction, which will increase
the numerous biotin signals even further. This cycling of the
background staining.
Dextran backbone
Avidin-biotin enzyme complex
HRP enzyme
Primary antibody
HRP conjugated
streptavidin
Tissue
antigen
Biotinyl-tyramide
deposition
81
Fluorescyl-tyramide can replace biotinyl-tyramide to avoid endogenous biotin background. In this procedure peroxidase is
tion methods have typically increased sensitivity by at least 50fold or greater (15). As with any amplification method, background tends to increase along with signal.
Dextran backbone
Substrate and
cross-linker
Enzyme
Primary antibody
Secondary antibody
Tissue
antigen
STEP 1
STEP 2
STEP 3
Red
ed chromogen
ch
AP conjugated
F(Ab) antibody
STEP 4
STEP
P5
Figure 6.6 Improved CSA system (iCSA). A proprietary methodology developed by Dako.
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expressed in benign prostate tissue. Thus it is used as a positive cancer marker, often in a multiplex stain with keratin and
dealing with small foci, with the result that some malignancies
may remain undiagnosed. In this context, multiple staining protocols significantly improve the ability to distinguish between
Technical Challenges
with the two basic colors. In the case where a rare target
problem.
topographic information.
83
Pre-treatment
Figure 6.7 Sequential double staining method performed with the EnVisionTM G|2 Doublestain Kit using polyclonal anti-kappa light chains (red)
and polyclonal anti-lambda light chains (brown) as primary antibodies.
FFPE tissue sections from tonsils.
classes:
Sequential staining
robust dyes such as DAB, Fast Red, AEC and Blue chromog-
Simultaneous staining
84
before the staining of the next target was performed. The dis-
DuoFLEX from
possible to mix reagents. Users will often find that the choice
TM
labeled with HRP and AP, respectively. Finally, the chromogens are applied sequentially. The result is a double stain
DAB and the primary rabbit antibodies are stained red with
ry antibodies with a biotinylated anti-mouse Fab fragment, followed by blocking of the remaining reagent with normal mouse
Multi-step technique
only small amounts of primary antibody are needed and the kit
flow cytometry. It essentially uses the same technique and offers la-
ed. The staining ends with the two enzymatic reactions being
performed sequentially.
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individual experiment.
Chromogenic Dyes
Enzyme
Chromogen
Color
Gal
X-Gal
Turquoise
ures 6.5 and 6.6). The method can bring low expressed targets
AP
Fast Blue BB
Blue
HRP
AEC
Red
HRP
DAB
Brown
Gal
X-Gal
Turquoise
AP
Red
HRP
DAB
Brown
AP
New Fucsin
Red
HRP
TMB
Green
Gal (beta-galactosidase); X-Gal (5-bromo-4-chloro-3-indolyl -galactoside); AP (alkaline phosphatase); HRP (horseradish peroxidase);
AEC (3-amino-9-ethylcarbazole); DAB (3,3-diaminobenzidine); TMB
(3,3,5,5-tetramethylbenzidine)
Fluorescent Dyes
Double immunofluorescence labeling is quite well established (22). Some of the same considerations as for chromogenic dyes apply when working with immunofluorescence.
86
Alternative dyes
and their variants), and Quantum dots. The latter, especially, has
to bind to the antigen of interest, which allows for antigen detection through fluorescence techniques. The degree of fluo-
because it does not rely on the human eye for detection and
differentiation. A digital image is acquired at excitation wavelengths relevant for the dyes applied, and separate detectors re-
cord individual colors. Thus, digital image analysis will allow the
combination of both fluorescent and immunoenzyme dyes (25).
Pros
Direct Immunofluorescence
Indirect Immunofluorescence
Simpler
used in image analysis should be optimized for the best fit possible with the detectors filter properties.
Image analysis systems incorporate algorithms that allow
Low costs
Cons
Lower signal
More steps
Higher costs
Less flexibility
87
Principle of Fluorescence
Fluorescence and phosphorescence are both types of lu-
Excitation decay
energy (heat) lost towards
semi-stable state
High energy
Excitation
by external
light source
Light emission
when the molecule goes
back to ground state
Low energy
The half-life of the excited state is generally less than 10 seconds. The electron loses a small amount of energy as heat,
and the remainder of the extra energy is given off in the form
Figure 6.9 Cultured pulmonary artery endothelial cells stained for tubulin (red), actin (green) and DNA (blue). The dual immunofluorescence procedure used rabbit-anti-actin and mouse-anti-alpha tubulin as primary antibodies. The secondary antibodies used were Texas Red-conjugated
goat, anti-rabbit IgG and FITC-conjugated goat, anti-mouse IgG. The sample was also stained with the DNA-specific dye Hoechst 33342. Scale
bar is equal to 20 microns.
88
light from fluorescein ranges from 490 nm to 630 nm, and the
Some fluorochromes have a small Stokes shift, while other fluorescent compounds have large Stokes shifts. For example,
Autofluorescence
light, and its Stokes shift is only about 20 nm, which means
that the light emitted is green. This contrasts with another flu-
green light, but has a large Stokes shift and thus the light will
Photobleaching
Stroke
shift
100
Energy
Peak FITC emision, 520 nm
S3
80
S2
Intersystem crossing
60
S1
T1
40
Ground
energy state
S0
20
Absorption (blue)
0
400
Phophoresence
450
475
500
525
550
575
600
625
650
675
700
Wavelength (nm)
Figure 6.11 Excitation and emission spectrum of fluorescein. When fluorescein is excited at a wavelength other than its peak excitation (470
nm in this example), the shape of the emission curve (darker green)
remains the same, but the relative intensity is reduced. The efficiency
of the excitation at 470 nm is 45% of peak excitation.
89
Fluorescence Overlap
Applications of IF in Pathology
lapping signal or it will give a false level for one or more colors.
been developed.
Intensity (%)
100
Excitation
source
80
60
40
Overlap
20
FITC emision
0
400
425
450
475
PE emision
500
525
550
575
600
Wavelength (nm)
90
625
650
675
700
Acknowledgements
Sections, in whole or parts thereof, from the previous editions of
this Guidebook are used in the 6th edition. We sincerely thank
and acknowledge the contribution of the authors. Special acknowledgements to: Mark Key, J. Paul Robinson, Jennifer Sturgis,
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