Immunohistochemistry (IHC) is a
molecular examine that involves the use of antibodies to detect specific
proteins within tissues on microscope slides. These proteins
are visualized with a colored chromogen and viewed with a Bright field
microscope. IHC can be used to detect a single protein in tissues or
antibodies can be multiplexed to detect two or more proteins on one
slide. Multiplexing is a brilliant way to analysis how proteins are cooperating
together within the paraffin embedded tissue or to view several different cell
populations at once.
The first and most important step in
any histology procedures, including IHC, is fixation. Typically, tissues are
fixed in 10% Neutral Buffered Formalin (NBF), processed and paraffin embedding;
formalin-fixed, paraffin-embedded tissue. The length of time tissues are in
fixative isn’t critical for routine H&E and special staining procedures;
however, it is critical for IHC. Formalin causes cross-linking within the
proteins of tissues; these cross-links can mask the epitopes which need to be
available to bind to antibodies in IHC procedures. Over-fixation can
render the tissues unusable for IHC. Ideally, paraffin embedded tissues
for IHC should be fixed for 24-48 hours, and then transferred to 70% ethanol
until processing, paraffin embedding and sectioning.
Although over-fixation of paraffin embedded tissues can result in un-breakable cross-linking within proteins in
tissue, optimal formalin fixation will also form cross-links. These
cross-links can be removed with a procedure called antigen (epitope)
retrieval. The two most common methods of antigen retrieval involve the
use of heat or enzymes. Heat-induced epitope retrieval (HIER) involves
superheating the slides to temperatures of 90°C-125°C for up to an hour.
HIER is done by placing deparaffinized and hydrated slides are placed in a
buffered solution which is then heated up in a pressure cooker, microwave or
steamer. Enzyme-induced epitope retrieval (EIER) is generally performed
on hydrated paraffin embedded tissue sections at either room temperature or at
37°C for 5-30 minutes.
Once retrieval procedures are
completed, the tissues are blocked for endogenous tissue elements which can
interfere with staining or cause background staining. These endogenous
elements can include: proteins, avidin, biotin and peroxidase. Once
blocking is completed, the antibody is applied to the tissue.
Next, antibodies are applied to the paraffin embedded tissue sections. Irrelevant control antibodies of the same
immunoglobulin isotype as the primary antibody are applied to a parallel set of
slides. For example, if the primary antibody is a Mouse anti-CD3, isotype
IgG2a; the irrelevant control antibody is Mouse IgG2a. The irrelevant
control antibody is applied at the same concentration as the primary antibody.
The isotype control antibody controls for non-specific binding of antibody to
the test tissue. The antibodies are incubated on the slides for 30-120 minutes
at room temperature, or overnight at 2-8°C.
The next step is antibody
detection. Detection is the step in which the antibody is bound to a
reagent that will react with a chromogen. The detection is usually done
with a secondary antibody that is labeled with an HRP (horseradish peroxidase)
polymer. If the primary antibody is a mouse, than the detection step
could be a Goat anti-Mouse HRP polymer. The polymer is generally applied
to the slide for 30-60 minutes.
After the detection step, the
colored chromogen is added, and the slides are counter stained. The most
common substrate chromogen is DAB (diaminobenzidine), which produces a brown
color when catalyzed by HRP. Thus, the protein or cell of interest will
appear brown on the slide. Although brown DAB is the most common, there
are other colors available; such as purple, pink/red, blue, green and
black. The slides are then counter stained with Hematoxylin, which stains
the nuclei a blueish-purple. Slides are then dehydrated in ethanol,
cleared in xylene and cover slipped with resinous mounting medium.
If you want to know more about
paraffin embedding, visit our site immunostaining. Here, our team of medical
experts will give you all brief history.
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