ENZYME

LABELING
OLIVE R. BAUTISTA ANDREA RAMOS
KARL BRYAN DAVE L. OLAR RICHELLYN VALERA
LEIGH ANN SUGUI JENNY LLARINAS
CJ ANGELO GAPASIN EMELY CHIGWAY

Enzymes are widely used in immunohistochemistry, and

are usually incubated with a chromogen using standard
histochemical method to produce a stable colored reaction
Enzyme labeling of antibodies with horseradish peroxidase,
followed by staining with appropriate substrate or chromogen
mixture such as diaminobenzidine (DAB), will produce an insoluble
dark brown reaction end product, and allow labeled cells to be
counterstained with hematoxylin and other nuclear stains

The optimal incubation time for linking antibodies with

peroxidase conjugates is 30 to 60 minutes at room temperature.
Different types of labels may be used for this type of biological
tagging. When an enzyme is chemically bound to another
molecule, the process is referred to as enzyme-labeling.
Enzyme-labeling may also be referred to as enzyme tagging.
 DIFFERENT TECHNIQUES/
METHODS
DIRECT
ENCHANCED POLYMER ONE STEP STAINING
TECHNIQUE
o conjugate the primary
antibody directly to the
label, such as a
fluorochrome or horseradish
peroxidase
o ADVANTAGE
-simple and quick
o DISADVANTAGE
-less sensitive
o The method is no longer
sufficiently sensitive for
today's demands
(EPOS) METHOD
o marketed by Dako A/S
o new direct technique whereby a large
number of primary antibody molecules
and peroxidase enzymes are attached to a
dextran polymer "backbone" or "spine
molecule"
o ADVANTAGE
§reduced number of incubation steps of
the staining protocol required
§more sensitive than the traditional direct
technique,
§suitable for frozen section
immunohistochemistry
o DISADVANTAGE
§limited number of primary antibodies
commercially available for this system
General EPOS Procedure (Peroxidase)
1. Quench for endogenous
peroxidase activity (optional).
2. Rinse with and place in wash
buffer for 3 to 5 minutes.
3. Incubate with EPOS conjugate for
10 to 60 minutes.
4. Rinse with and place in wash
buffer for 3 to 5 minutes.
5. Incubate with substrate-
chromogen for 5 to 15 minutes.
6. Counterstain (optional) and
coverslip.
 DIFFERENT TECHNIQUES/
METHODS
INDIRECT

THREE-STEP
TWO STEP INDIRECT
INDIRECT
TECHNIQUE TECHNIQUE
TECHNIQUE
o 2 or 3 step procedure o unconjugated primary antibody o second enzyme-
first binds to the antigen
o ADVANTAGE o enzyme-labeled secondary
conjugated antibody is
§relatively inexpensive antibody directed against the added to the previously
primary antibody (now the described indirect
§more sensitive antigen) is then applied, followed
by the substratechromogen
technique
o Horseradish solution o addition of a third layer
peroxidase is the most o primary antibody is made in of antibody serves to
rabbit or mouse, the secondary
commonly used antibody must be directed against
further amplify the signal,
enzyme for indirect rabbit or mouse immunoglobulins since more antibodies are
antibody enzyme- o Cross-reactivity is eliminated by capable of binding to the
using preabsorbed secondary
complex techniques antiserum
previously bound
secondary reagent
 Soluble Enzyme Immune Complex
Techniques (Unlabeled Antibody Techniques)

o utilize preformed soluble enzyme-anti-enzyme immune complex

o staining sequence involves the use of an unconjugated primary
antibody, a secondary antibody, the soluble enzyme-anti-enzyme
complex, and the substrate solution

·Immunohistochemistry (IHC) is a mainstay immunoassay

for many laboratories and is used to demonstrate the
presence or absence of important proteins, detect post-
translational modifications, diagnose disease, and more.
Rockland produces highly-active antibodies and conjugates
for use in IHC experiments. Rockland also produces high-
quality reagents for the setup and detection in IHC
experiments. Protect your experiments with Rockland
antibodies.

IHC uses both immunological and biochemical techniques to

examine distinct tissue sections by the use of labeled antibodies
interacting with target antigens. A visible label or stain is used to
visualize the antibody antigen interaction. IHC allows for the
visualization of distinctive cellular components of cells and is
frequently used by pathologists and in diagnostics.

IHC SAMPLE
PREPARATION
The antigen is fixed and adhered to a
glass microscope slide. Formaldehyde
fixation is suggested as the routine
initial method of choice for tissue and
cell fixation, but other methods are also
suitable. Background staining is reduced
with a blocking buffer to block non-
specific sites to which the antibodies
may bind. Rockland offers common
blocking buffers including BSA, normal
serum, non-fat dry milk, and an
optimized proprietary blocking buffer.
IHC SAMPLE LABELING
·Labeling is achieved by the reaction of a
primary antibody with the immobilized antigen
to form an antigen-antibody complex. A second
biotinylated antibody specific for the primary
antibody host species reacts with the complex.
Streptavidin conjugated to the enzyme
peroxidase or alkaline phosphatase reacts with
this complex. Finally, a substrate is added to
causes a reaction with the enzyme, producing
a colored precipitate to form on the slide at
the location of the antigen. The slide is viewed
through a standard light microscope and
photographed. A common manipulation in IHC
fixing is the use of the antigen retrieval
technique, a simple method of heating or
boiling paraffin-embedded tissue sections to
enhance the observed signal.
GROUP 4

Thank you!