HISTOPATHOLOGY

CGHMC - Laboratory
Ms. Janine Mae Quimbo

example, using specific tumor markers,

physicians use IHC to diagnose if a tumor is
benign or malignant, to determine its stage and
grade, and to identify the cell type and origin of
- Immunohistochemistry (IHC) combines
a metastasis in order to find the site of the
anatomical, immunological and biochemical
primary tumor. A variety of other non-neoplastic
techniques to image discrete components in
diseases and conditions are diagnosed using
tissues by using appropriately-labeled
IHC as a primary tool or as a confirmatory
antibodies to bind specifically to their target
procedure.
antigens in situ.
- In a research context, IHC can be used alone or
- antigen(proteins in the surface of the cell) and
in conjunction with other analytical techniques
antibody (stain) binding is incorporated into the
to study, for example, normal tissue and organ
method, successful binding/staining of the
development, pathological processes, wound
specific stain means positive result (ex. PD-L1
healing, cell death and repair, and many other
Immunostain = positive to lung carcinoma)
fields.
- IHC makes it possible to visualize and
- IHC is also used in drug development to test
document the high-resolution distribution and
drug efficacy by detecting either the activity or
localization of specific cellular components
the up- or down-regulation of disease markers
within cells and within their proper histological
in the target tissues and elsewhere.
context.
- Traditional IHC is based on the immunostaining
- Main goal: help and aid pathologist to diagnose
of thin sections of tissues attached to individual
the patient’s cases (carcinoma, tumor, other
glass slides. Multiple small sections can be
tissue related disease)
arranged on a single slide for comparative
- While there are multiple approaches and
analysis, a format referred to as a tissue
permutations in IHC methodology, all of the
microarray. (routine: cut px specimen, mount to
steps involved are separated into two groups:
the slide, h&e stain)
sample preparation and sample staining.
- Typically, IHC slides are prepared, processed,
and stained manually or in small groups.
History However, current technology provides
The principles of IHC have been known since the automation options for high-throughput sample
1930s, but it was not until 1942 that the first IHC preparation and staining. Samples can be
study was reported. Coons et al. (1942) used viewed by either light or fluorescence
FITC-labeled antibodies to identify Pneumococcal microscopy (In CGHMC, IHC staining is
antigens in infected tissue. Since then, major automated)
improvements have been made in tissue fixation
and sectioning methods, antigen/epitope retrieval,
antibody conjugation, immunostaining methods and
reagents, as well as microscopy itself. As a result, - While using the right antibodies to target the
IHC has become a routine, but essential tool in correct antigens and amplify the signal is crucial
diagnostic and research laboratories. for optimal visualization, complete preparation
of the sample is critical to maintain cell
Application morphology, tissue architecture and the
- IHC is used for disease diagnosis, biological antigenicity of target epitopes.
research, and in drug development. For
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HISTOPATHOLOGY
CGHMC - Laboratory
Ms. Janine Mae Quimbo
- Most tissue fixatives chemically crosslink
proteins and/or reduce protein solubility, which
can mask target antigens during prolonged or
improper fixation. Therefore, the right fixation
method must be optimized based on the
application and the target antigen to be stained.
- The most common fixative is formaldehyde
(formalin)[10% neutral buffered formalin], a
semi-reversible, covalent crosslinking reagent
that can be used for perfusion or immersion
fixation for any length of time, depending on the
level of fixation desired. Tissues fixed in
formaldehyde are typically embedded in paraffin
wax to permit sectioning and further processing.
Such tissues and the sections cut from them
are often referred to as formalin- fixed and
paraffin-embedded or FFPE.
- Formalin-fixed tissue samples are usually
embedded in paraffin to maintain their natural
shape and tissue architecture during long-term
storage and to facilitate sectioning prior to IHC.
Such samples and the sections prepared from
them are usually referred to as formalin- fixed,
paraffin-embedded (FFPE) materials.
- Biopsy/Routine process → Read and
diagnosed by pathologist → Doctor request IHC
staining
- FFPE tissues are usually cut into sections as
thin as 4 to 5 µm with a microtome.
- These sections are then mounted onto glass
slides that are coated with a tissue adhesive.
- Charged slides are used in IHC for better
adherence of tissue on the slides
- This adhesive is commonly added by
surface-treating glass slides with 3-
aminopropyltriethoxysilane (APTS) or
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poly-L-lysine, both of which leave amino groups
on the surface of the glass to which the tissue
adheres.
- In the past, and now, if necessary, slides can be
coated with actual adhesives, including gelatin,
egg albumin or even Elmer's glue. After
mounting, the sections are dried in an oven or
microwaved in preparation for
de-paraffinization.
- The paraffin in FFPE sections must be
completely removed before IHC staining. If
de-paraffinization is not complete, the target
antigens will be obscured and the antibodies
will be unable to react with them. In fact,
paraffin's hydrophobicity actually repels
aqueous solutions containing the IHC staining
reagents.
- Paraffin is hydrophobic and repels aqueous
solutions (reagents used in IHC are
aqueous)
- Flammable, toxic, and volatile organic solvent
xylene has traditionally been used to
de-paraffinize FFPE slides, although
xylene-free de-waxing alternatives are now
available. (in the lab xylene is used)
- Formaldehyde fixation generates methylene
bridges that covalently crosslink proteins in
tissue samples. These bridges can mask
antigen and/or epitope accessibility and inhibit
or prevent antibody binding. As a result, FFPE
sections typically require treatment designed to
unmask or retrieve the antigenic epitopes prior
to staining. This is called epitope or antigen
retrieval.
- Epitope/antigen retrieval is usually performed
by heating or boiling the de- paraffinized
sections in various buffers at different pH
values, which is called heat-induced epitope
retrieval or HIER. Antigens can also be
HISTOPATHOLOGY
CGHMC - Laboratory
Ms. Janine Mae Quimbo

retrieved by digesting the tissue sections with a

proteolytic enzyme like pepsin, trypsin, or
proteinase K.
- Counterstains provide contrast to the primary
- DAKO Autostainer is where the slides are
stain and can be cell structure-specific. These
treated first, it also has high or low pH
single-step stains are usually added after
- Cytokeratin autostainer machine needs
antibody staining. Common counterstains
protease
include hematoxylin, eosin, nuclear fast red,
methyl green, DAPI, and Hoechst fluorescent
stain.

- Although antibodies show preferential avidity

and affinity for specific epitopes, antibodies may
partially or weakly bind nonspecifically to sites
- After all staining is completed, the sample
on non-antigen proteins that mimic the correct
should be preserved for archiving purposes and
binding sites on the target antigen.
to prevent enzymatic product solubilization or
- In the context of antibody-mediated antigen
fluorophore photobleaching. Sealing the sample
detection, nonspecific binding causes high
by mounting a coverslip with an appropriate
background staining that can mask the
mounting solution (mountant) stabilizes the
detection of the target antigen.
tissue section and the stain.
- To reduce background staining in IHC, ICC, and
any other immunostaining application, prior to
staining, the samples are incubated with a
buffer(always fresh and new, replaced after 1 - Once the sections are prepared, the samples
week) that blocks the non-specific sites to which are viewed by light or fluorescence microscopy.
the primary or secondary antibodies may Depending on the antibody detection method,
otherwise bind. one can perform confocal microscopy for
greater detail and enhanced imaging
capabilities.

- Detecting the target antigen with antibodies is a

multi-step process that requires optimization at
every level to maximize signal detection.
- The machine works with barcode which
means it will not put reagent on a slide if it is
not needed
- Make sure labeling is correct as well as the
barcode
- Both primary and secondary antibodies are
diluted into a buffer formulated to help stabilize
the antibody, promote its uniform and complete
diffusion into the sample, and discourage
nonspecific binding.
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