Immunohistochemistry (IHC) is a technique that combines immunology and histology to detect antigens in tissues using antibodies, with the main steps including preparing tissue sections, using primary and secondary antibodies to target antigens, developing signals using reporters and counterstains, and observing under a microscope. IHC is widely used for disease diagnosis by identifying molecular markers characteristic of cellular events involved in diseases like cancer and neurological disorders. Troubleshooting for IHC focuses on reducing background staining and improving antigen detection through optimizing antibody concentrations and incubation times.

1. An Introduction to
Immunohistochemistry

2. What is Immunohistochemistry?
 Histochemistry is a science that combines the
techniques of biochemistry and histology in the
study of the chemical constitution of tissues and
cells.
 Immunology is a science that deals with the
immune system, cell-mediated and humoral
aspects of immunity and immune responses
 Immunohistochemistry (IHC) is the
localization of a known antigen in tissues by
utilizing antibodies directed towards that
(specific) antigen
Introduction

3. STEPS
6
Secondary Antibody
5
Primary Antibody
4
Blocking Background
Staining
3
Blocking Endogenous
Enzymes
2
Antigen Retrieval
1
Tissue Sections
Chromogen Substrate Counterstain Mounting
7 8 9
Mircoscopy Observation

4. Sample
Preparation01

5. 回顾
Fixation
 The fixation can help 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
 Most of the Ab available are optimized for use with formaldehyde

6. 回顾
 The tissue may then be sliced or used whole, dependent upon the purpose of
the experiment or the tissue itself.
 Specimens are typically sliced at a range of 3 µm-5 μm. Then they are further
adhered to the slides by baking at 60℃
 Deparaffinization：The slices are then mounted on slides, dehydrated using
alcohol washes of increasing concentrations (e.g., 50%, 75%, 90%, 95%, 100%),
and cleared using a detergent like xylene before being imaged under a
microscope.
Preparing Tissue Slices
Note：Due to the different methods of fixation and tissue preservation, the
sample may require additional steps to make the epitopes available for
antibody binding.

7. 回顾
 A great amount of non-specific binding causes high background staining which
will mask the detection of the target antigen.
 In order to reduce background staining in IHC, samples are incubated with
blocking buffers, including normal serum, non-fat dry milk, BSA, or gelatin.
 In addition，many methods can have a try：
· Dilution of the primary or secondary antibodies,
· Changing the time or temperature of incubation
· Using a different detection system or different primary antibody.
Reducing Non-specific
Immuno-staining

8. Preparation Protocol
Tissue Fixation
v
Creative
Bioarray
Tissue Slices & Antigen Retrieval
v
Creative
Bioarray
Blocks All Non Specific Sites
Peroxide Block Protein Block

9. Sample
Labeling02

10. Antibody Types
• Polyclonal antibodies
• Monoclonal antibodies
• Primary antibodies
• Second antibodies
• With chromogenic
reporters, an enzyme label
reacts with a substrate to
yield an intensely colored
product that can be
analyzed with an ordinary
light microscope.
• Direct method
• Indirect method
• After immunohistochemical
staining of the target
antigen, a second stain is
often applied to provide
contrast that helps the
primary stain stand out.
IHC Reporters Target Antigen
Detection Methods
Counterstains

11. • The antibodies used for specific detection can be polyclonal or monoclonal :
Polyclonal antibodies are a heterogeneous mix of antibodies that recognize several epitopes.
Monoclonal antibodies show specificity for a single epitope.
• For immunohistochemical detection strategies, antibodies are classified as
primary or secondary reagents:
Primary antibodies are raised against an antigen of interest and are typically unconjugated
(unlabeled), while secondary antibodies are raised against immunoglobulins of the primary antibody
species.
The secondary antibody is usually conjugated to a linker molecule, such as biotin, that then
recruits reporter molecules, or the secondary antibody itself is directly bound to the reporter
molecule.
Antibody Types

12.  The most popular being chromogenic and fluorescence detection mediated by an
enzyme or a fluorophore, respectively.
 Alkaline phosphatase (AP) and horseradish peroxidase (HRP) are the two
enzymes used most extensively as labels for protein detection.
 Fluorescent reporters are small, organic molecules used for IHC detection and
traditionally include FITC, TRITC and AMCA.
IHC Reporters
 For chromogenic and fluorescent detection methods, densitometric analysis of the
signal can provide semi- and fully quantitative data, respectively, to correlate the
level of reporter signal to the level of protein expression or localization.

13. Target Antigen Detection Methods
 The direct method is a one-step staining method and involves a labeled
antibody (e.g. FITC-conjugated antiserum) reacting directly with the
antigen in tissue sections. While this technique utilizes only one antibody
and therefore is simple and rapid, the sensitivity is lower due to little signal
amplification, in contrast to indirect approaches.

14. Target Antigen Detection Methods
 The indirect method involves an unlabeled primary antibody (first layer)
that binds to the target antigen in the tissue and a labeled secondary
antibody (second layer) that reacts with the primary antibody.
 As mentioned above, the secondary antibody must be raised against the
IgG of the animal species in which the primary antibody has been raised.
This method is more sensitive than direct detection strategies because of
signal amplification due to the binding of several secondary antibodies to
each primary antibody if the secondary antibody is conjugated to the
fluorescent or enzyme reporter.

15. Counterstains
 After immunohistochemical staining of the target antigen, a second stain
is often applied to provide contrast that helps the primary stain stand out.
Many of these stains show specificity for specific classes of biomolecules,
while others will stain the whole cell.
 Both chromogenic and fluorescent dyes are available for IHC to provide a
vast array of reagents to fit every experimental design, and include:
hematoxylin, Hoechst stain and DAPI are commonly used.

16. Troubleshooting
03

17. Strong Background Staining
• Endogenous biotin or reporter enzymes or
primary/secondary antibody cross-
reactivity are common causes of strong
background staining
Weak Target Antigen Staining
• Weak staining may be caused by poor
enzyme activity or primary antibody
potency
Autofluorescence
• Autofluorescence may be due to the
nature of the tissue or the fixation method

18. Reduce incubation time
Reduce incubation temperature
Reduce substrate incubation time
Reduce antibody concentration or perform a
titration to determine the optimal dilution for
primary and secondary antibodies
The concentratin of antibodies was too high
Incubation time was too long
Incubation temperature was too high
Substrate incubation time was too long
Sections dried out Avoid sections being dried out
Troubleshooting: Strong Background Staining
Sources Solutions

19. Replace with a new batch of antibodies
Aliquot antibodies into smaller volumes and store in
freezer(-20 to -70℃) and avoid repeated freeze and
thaw cycles
Increase the concentration of antibodies. Or run a
serial dilution test to determine the potimal dilution
that gives the best signal to noise ratio
Deparaffinize sections longer or change
fresh xylene
Indequate deparaffinization
Inactive primary antibodies
Antibodies do not work due to improper
storage
Antibody concentration was too low
Inadequate antibody incubation time Increase antibody incubation time
Troubleshooting: Weak Target Antigen Staining
Sources Solutions
Inadequate or improper tissue fixation
Increase duration of post fixation or try different
fixatives

20. If the fixation step is the cause of the autofluorescence, test different
fixatives (i.e., if aldehyde fixation is used, try a non-aldehyde fixative)
to determine if autofluorescence can be reduced without sacrificing
antigen detection.
If aldehyde fixation is used and no other fixative can be used, then
fixative-induced autofluorescence may be reduced by treating the
sample with ice cold sodium borohydride (1 mg/mL) in PBS or TBS.
If there is autofluorescence in the test sample, then this suggests that
either the tissue sample shows inherent autofluorescence (which is
common) or that the fixation method is causing the sample to
autofluoresce.
Troubleshooting: Autofluorescence
Detection Methods
Treat the tissue sample with dyes that quench fluorescence. These dyes
include:
Pontamine sky blue, Sudan black, Trypan blue, Paraffin-embedded

21. Diagnosis
of
Diseases
Characteristic
of Particular
Cellular Events
Basic
Research
Histology
Research
Applications
It is widely used in the diagnosis of diseases including cancer, neurological disease, digestive disease, etc.
Specific molecular markers have been found and verified to be characteristic of particular cellular events in
disease such as proliferation, apoptosis and inflammation. Thus it could be used in detection of such changes
involved in disease. IHC is also a common method in basic research of disease to help understanding the
distribution and localization of biomarkers and differentially expression of proteins in different parts of tissue.

22. Creative Bioarray will give you the best and most
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23.  Frozen Embedding and Sectioning
 Paraffin Embedding and Sectioning
 Cell Pellet Embedding and Sectioning
Start Program
Preparation of Tissue Sections
 H&E Staining
 Ki67+ (Cell proliferation)
 TUNEL (Cell apoptosis)
 CD15 (Hodgkin’s Disease)
 CD117 (Gastrointestinal stromal tumors)
 Cytokeratins (Carcinomas)
 CD10 (Renal cell carcinoma and acute
lymphoblastic leukemia)
 CD20 (B-cell lymphomas)
 CD3 (T-cell lymphomas)
 Matrix metalloproteinase 9 (Carcinomas)
 PCNA (Carcinomas)
 CA-15-3 (Breast Tumor)
Selection of Antibodies
Staining
 Pathological evaluation including scoring
and analysis
 High-resolution digital imaging
 Spot counting and other image analysis
 Pathology consultation
Report and Analysis

24. For more info please contact us:
E-mail: info@creative-bioarray.com
Go to our website：
www. creative-bioarray.com