CST Guide:

12
Section II: ANTIBODY APPLICATIONS
chapter
Antibodies targeting
the same protein
may have different
preferred methods
of antigen retrieval.
EGF Receptor (D38B1) XP ® Rabbit
mAb #4267: IHC analysis of paraffinembedded
human lung carcinoma
using #4267 and an EGFR mouse
mAb after antigen retrieval by boiling
in citrate buffer (left), boiling in EDTA
buffer (center), or digestion with
pepsin (right).
For #4267, superior signal is
obtained with EDTA retrieval.
However, for the competitor’s EGFR
mouse mAb, signal is only achieved
with pepsin digestion.
Immunohistochemistry (IHC)
Tissue Analysis
Immunohistochemistry (IHC) is a common technique for morphological characterization of tumors or other
tissue malignancies. IHC uses antibodies to detect and analyze protein expression while maintaining
the composition, cellular characteristics, and structure of native tissue. Starting with high quality tissue
samples is important for successful IHC results, so it is best to use freshly cut sections. It is also essential
to include appropriate controls to assess antibody performance. Prior to testing on tissue, antibody
specificity can be evaluated at the cellular level using a variety of cell lines and treatment conditions.
• Total protein specificity can be assessed through the use of positively and negatively expressing
cell lines.
• Cells can be treated with biological or chemical modulators known to induce signaling changes
to verify modification specificity, such as phosphorylation, acetylation, cleavage, etc.
• Phospho-specific antibodies can be evaluated with phosphatase treatment.
• Isotype control antibodies help rule out nonspecific staining of primary antibodies due to Fc
receptor binding or other protein-protein interactions and should have the same immunoglobulin
type as the test antibody.
The IHC protocol includes variations in antigen retrieval methods, antibody diluents, and detection
systems; the optimal method for these parameters must be determined experimentally for each
primary antibody in order to produce the maximum signal.
The most common problems encountered when performing IHC are weak specific staining or high
background. Some challenges, such as incomplete or poor antigen retrieval, are broadly applicable to
most antibodies, while other challenges are specific to certain species or protein modifications.
IHC Tips for Success
Antigen retrieval optimization can greatly improve staining.
The crosslinks created during the fixation step can prevent antibody binding by inhibiting access to the
antigen; therefore, it is important to reverse crosslinks using a procedure called antigen retrieval (also
known as antigen unmasking or epitope retrieval). Antigen retrieval can be achieved through either
a heat-induced method (heat-induced epitope retrieval; HIER) or through enzymatic digestion, and
the optimal form of retrieval is specific for the unique antibody/antigen and not for the protein itself.
Therefore, if you use more than one antibody against a particular protein target, the optimal retrieval
conditions for each antibody must be determined individually.
CST #4267
Competitor’s mAb
Citrate EDTA Pepsin
Avoid casein-based solutions when
using a phospho-specific antibody.
Blocking is an important step in the IHC protocol because it prevents nonspecific background staining.
Commercially available blocking solutions that contain casein should be avoided when working with
phospho-specific primary antibodies as they tend to diminish signal intensity.
Phospho-Histone H2A.X (Ser139)
(20E3) Rabbit mAb #9718: IHC
analysis of paraffin-embedded human
breast carcinoma using #9718 after
blocking with normal goat serum
(NGS) (left) or a casein-based blocking
solution (right).
TBST/5% NGS
Avoid same-species background.
Casein
When the primary antibody is from the same species as the sample being tested, the secondary antibody
may bind endogenous IgG in some tissues, causing high background (mouse-on-mouse staining,
for example). Including a control slide stained without the primary antibody can establish whether or
not the secondary antibody is the source of the background.
Ki-67 (D3B5) Rabbit mAb (Mouse
Specific; IHC Formulated) #12202:
IHC analysis of paraffin-embedded
mouse lung using #12202 (left) or
a competitor’s Ki-67 mouse mAb
(right). Analysis with #12202 displays
only nuclear staining, while analysis
with the mouse mAb shows high
background staining.
CST #12202
Competitor’s mouse mAb
Use a polymer-based detection system to increase sensitivity
and avoid endogenous biotin background staining.
Traditional IHC detection methods use a biotinylated secondary antibody followed by exposure to an
avidin-HRP complex prior to chromogenic detection. Biotin-based systems are prone to background
staining, particularly in tissues that possess high levels of endogenous biotin, such as liver and kidney.
Polymer-based detection reagents consist of enzymes and secondary antibodies directly conjugated to
a polymer backbone. These systems eliminate false-positive staining due to endogenous biotin. The
polymer-based system also improves sensitivity, enabling detection of low abundance targets. Signal-
Stain ® Boost IHC Detection Reagents save time by eliminating one step in the detection procedure and
are compatible with all peroxidase-based substrates.
SignalStain ® Boost IHC Detection
Reagent #8114: IHC analysis of
paraffin-embedded human lung carcinoma
using Sox2 (D6D9) XP ® Rabbit
mAb #3579 and either biotin-based
detection (left) or polymer-based
detection #8114 (right).
Competitor antibodies used in accordance with manufacturer recommendations.
Biotin-based Detection
Polymer-based Detection
12: Immunohistochemistry (IHC)
Casein block
produces a lower
overall signal when
compared with
TBST/5% NGS.
When possible,
avoid using primary
antibodies raised in
the same species as
the tissue examined.
Polymer-based
detection is more
sensitive than biotinbased
systems.
Competitor antibodies used in accordance with manufacturer recommendations.
214 For Research Use Only. Not For Use in Diagnostic Procedures. See pages 302 & 303 for Pathway Diagrams, Application, and Reactivity keys.
www.cellsignal.com/cstihc
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