Immunohistochemistry (IHC) Principle

Immunohistochemistry (IHC) Principle
Adetailed presentation ofImmunohistochemistry principle.

 Immunohistochemistry (IHC) is a method that combines biochemical, histological andimmunological techniques intoa simple but powerful
assay forprotein detection. IHC provides valuable information as it visualizes the distribution andlocalization ofspecific cellular components
within cells andin proper tissue context.
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What is Immunohistochemistry (IHC)?

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Immunohistochemistry Principle
 In order tobe abletouse certain antibodies, samples must be
processed orfixed in a very specific manner. For example, many
antibodies only recognize proteins that have been reduced and
denatured, whileothers only detect protein epitopes when the
proteins arein their native, folded conformation. Some
antibodies should only be used with unfixed, frozen tissue. Many
antibodies cannot bindto their targetepitopes in formalin-fixed,
paraffin-embedded tissues unless an antigen retrieval step is
included toreverse the cross-links that result fromfixation.
 If yourprotein ofinterest is intracellular, cells must be
permeabilized with solvents ordetergents toallow antibodies
access tothe interior ofthe cell. Detergents arethe most popular
method forpermeabilizing cells. However, because they disrupt
the membrane, detergents arenot appropriateforcytoskeletal
antigens orlow molecular-weight targets such as viralantigens
andsome enzymes. Note that permeabilization is necessary for
ICC.For IHC (Paraffin and frozen sections), the tissue is usually
sliced thinly, and thus permeabilization is not required.

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Stepwise IHC Process
Fixation
Tissue Sectioning
Paraffin Embedding
Inactivation
Blocking
Antigen Retrieval
Detection
Chromogens, Counterstains andMounting Media

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Whyis Fixation important?
 Fixation preserves morphology, prevents tissue degradation,
ensures antigenicity andhelps protect cells fromdamage during
the strenuous tissue preparation process. The reproducibility and
quality ofIHC results aredependent on fixation times,
temperatures, pH andbuffer composition — allofwhich must
be carefully optimized andstandardized by the experimenter.
Fixation
A.The Purposeof Fixation
 Keepcellsharpandtissueshapetopreventpostmortemautolysis,
putridness,endogenicand exogenicenzyme activity.
 Maintain cellstructureand positionbypreventingantigendiffusion
throughtransferofprotein,fat, sugarandenzymes ofcellintoinsoluble
substances.
 Precipitateand curdlematerialsintissuetoproducedifferentrefraction.
 Induratetissuestoenhanceworking withglassslides.
 Preventcellfrom shrinkingandswelling.
 Give colortoclarifytissuesby differentaffinitytocoloringagent.

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B. Selectionof Fixing Solution
 Below is a list ofcommonly used fixing solutions. You may need
totest whether a specific type ofsolution is appropriateforyour
detected antigens because there is no standardfixing solution for
different kinds of antigen immobilization.
Fixation
I. Acetone andAlcohol
 These two types ofsolutions, which areprimaryfixing solutions,
play arole ofprecipitating sugars andfat as well as maintain the
immunologic competence.
 Alcohol is ineffective tomaintain low molecular weight protein,
polypeptide andcytoplasmic proteins. However, it can be mixed
with glacial acetic acid, ethyl ether, chloroform and formaldehyde.
 Acetone is often used forfrozen tissue andcytological smears
because it has astrong penetrability anddehydration property.

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II. Aldehyde
 It isadi-functional cross-linkingagentwhichiswidelyusedduetoits
strongpenetrability,lowcontractibilityandlowbackground.Ithelps
keep the cross-linkingbetweentissuesand maintainantigen.
 Formalin(10% neutralbuffered)isthe mostwidelyused
 4% paraformaldehydeisbetterthan formaldehyde
 Bouin’s solution(containing picricacid)isthe mostwidelyusedin
histologyandpathology
 Zamboni’s solutionisapplied to lightand electronmicroscopic
immunocytochemistryand isbetterthan formaldehydein
ultrastructuralorganizationmaintenance
Fixation
III. Non - Aldehyde
 Carbodiimide, dimethylacetamide, dimethyl-suberimidate, para-
benzoquinone arewidely used in tissue fixation ofpeptide
hormones
 These fixation agents arebetter mixed with glutaric dialdehyde
orparaformaldehyde
 In recent years,a new type offormaldehyde-free fixing solution
has become available. With lowtoxicity anddegradable chemical
agent, this solution has gained a broadpopularity in IHC, regular
pathological examinations andmolecular pathology detections
due tothe use ofnon-protein cross linking, strong DNA/RNA
preservation andabsence ofcell vacuole, tissue shrinkage and
pyknosis.

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I.Fixation byPerfusion
 The best waytopreserve tissue morphologyandtarget protein
antigenicity is toreplace the animal’s entire systemic blood
volume by fixative with vascular perfusion. This is followed by
perfusion with sucrose solution. It is important toremember that
fixation can cause cross-linking which masks epitopes.
Fixation : Fixationmethods
II. Fixation byImmersion
 It is sometimes impractical orinappropriatetofix tissues by
perfusate. An alternative method is toimmerse tissues in fixative
solution (at 4°Cif needed) for a specified period which is
determined by the antigen stability andtype of fixing solution
used. Biopsy andsurgical specimens as well as other non-
irrigationtissues commonly employ this fixation method.

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III. SnapFreezing
 It is not always possible orappropriatetofix tissues before
sectioning. For example, phosphorylation-dependent epitopes
have been shown totranslocate fromthe membrane tothe
cytoplasm following fixation with formaldehyde. For cases in
which standardfixation techniques cannot beused orfixation
will be carried out later, tissue can be snapfrozen andkept at -
70°Cuntil use.
Fixation : FixationMethods
Exercise cautionwhenfixating tissues
 Donot over-fix the tissues
 Keep the tissues fresh after fixation
 Use enough fixing solution andwash it off completely after
fixation
 Use tissues ofsize less than 2 cm ×1.5 cm ×0.3 cm (Thickness <
0.3 cm)

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A.Slide Pre-Treatment
 The following pre-treatment procedure is designed toprevent
peeling caused by elevated temperature, high pressure, radiation
andother factors.
 They aredifferent pre treatment procedures for different section
types ;
 I. Microscopic Slide
 II. Cell samples
 III. Tissue Section mounting
Tissue Sectioning
MicroscopicSlide
 Due tothe oil attached on surface, anew slide should bedipped
intocleaning solution for 12 to24 hours. After washing the slide
morethan 5X in distilled water, dip it into 95% alcohol for 2
hoursfollowed bydrying with simple wiping orin an infrared
oven. Pay attention toavoid scratching the slide duringwashing.
 Note: Microscopic slide for IHC is required tobe 5 μm.However,
the slide fornervous tissue should be 20-100 μmtoenhance the
tracking ofnerve fiber direction.

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(ii)Cell Samples
(a) Adherent Cells
 Cell Climbing: Growadherent cells on multi-aperture culture
plates with coverslip*, culture vessels orchamber slide
 Direct Cell Culture: Cultureadherent cells directly on culture
vessels or multi-aperture culture plates
* The coverslip pre-treatment procedure is very similarto the one
forthe microscopic slide except that dipping andcleaning should
be completed in 2 hours because the coverslip is much thinner)
Tissue Sectioning
(b) Non-AdherentCells
 Cell Smear: Adhere non-adherent cells on coverslip with
chemical bond
 Eccentric Cell Smears: Adhere non-adherent cells on culture
vessels by cell micro-centrifuge

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(iii) Tissue Section Mounting
 Dilute 3-Amino Propyl Tri-ethoxy Silane (APES) by acetone
 Coatthe microscopic slide with APES
 Apply tissue on the coated slide followed by adding a dropof
mounting medium (Glycerol Gelatin) on the tissue
 Hold the coverslip at45°, allowing the droptospread along the
edge ofthe slip
 Slowly cover the tissue entirely with the coverslip
 Incubate the slide at 80°Cfor 1 hourif there is an adhering
problem with a tissue section
Tissue Sectioning
(B) Tissue Section Types
 Tissue samples aretypically taken from specimens ofvarious
sources: biopsy, surgery,animal model andautopsy. The first
three types ofspecimens give fresh tissues while the lastone
(autopsy) is taken after an animal has died fortwohours which is
moreor less apostmortem autolysis. As antigens may denature,
disappear anddiffuse, autopsy specimen should be fixated as
soon as possible so as not toinfluence its label.

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(i) Frozen
 The most important feature forthis type oftissue section is to
keep antigen’s immune competence completely, especially for
the cell surface antigen. Both fresh andfixed tissues can be
processed as frozen tissues. However, the tissues must be dried
(or primaryfixed) andstored at lowtemperature.
Tissue Sectioning
(ii) Paraffin-Embedded
 Paraffin-embedded tissue section is normallysliced by a rotary
microtome togivea thickness of2-7 μm. With proper treatment,
the section reveals clear tissue structure andexact antigen
location toenable high medical-value pathology researches and
retrospective studies. This section type can be stored at 4°Cfor
long term use.

 Use sharpknife andscissors to avoidextrusion damage
 Cutter should be flat,small and thin (Normal size is 1.0 cm ×1.0 cm ×0.2 cm)
 Eliminate fat tissue andcalcification zone
 Collect samples from live animals and fix samples immediately after wash
 Choose diseased instead of necrotic region
 Choose normal tissue as control if necessary
 Make paraffin-embedded tissue orfrozen tissue immediately after sectioning orstore the tissues in liquid nitrogen container or refrigerator
at -70°C
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Tips & Techniquewhencollecting, fixating and sectioning the samples

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(A) Dehydration
This step removes water completely, creates a condition forthe next
step andhardens the tissue ofinterest. The dehydrating agents
describes below arecompletely miscible with water andcan be
prepared in different volumetric ratioswith water.
(I) Ethanol
 As the most commonly used dehydrating agent, ethanol has a
strong water separation and tissue hardening capability.
However, since ethanol has strong penetration andcontractility,
its concentration should be progressively increased toavoid
tissue excessive shrinking.
Paraffin Embedding
(ii) Acetone
 As ausual substitute for alcohol, acetone acts as both a fixing
solution andfast dehydrating agent. Payattention tothe
dehydrating time with acetone as it tends toover harden tissues.

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Transparentizing
 Afterdehydration,thetissueofinterestrequiresa transparentizing
stepbecausethe dehydratingagent usedinthe preiousstepis
immisciblewiththe paraffinfrom oneofthe subsequentsteps. The
additionoftransparentreagenthelpsparaffinabsorbintothe tissue.
Commontransparentreagentsare:
(I)Xylene
 Asthe mostwidelyused transparentreagent,xylene ismisciblewith
bothethanoland acetone,anditactsasafusingagent forparaffin
wax. Sincexylene hasastrongandfast contractilitytotissue,the
tissueshouldnotbeimmersedforan extended time periodoritwill
be overcrispand too hard.
Paraffin Embedding
(ii) BenzeneandToluene
 These reagents aresimilar toxylene. However, they haveweak
andslow contractility totissue, andtherefore the tissue can be
immersed in these reagents fora longer time. Note that benzene
andtoluene have high toxicity andmust be handled with care.

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(iii) Chloroform
 Comparedtoxylene, benzene andtoluene, chloroform is a much
gentler reagent. However, it has asmall refractive index, andthe
tissue should thus be immersed in chloroform fora longer time
than the other transparent agents in order toguarantee complete
penetration.
Paraffin Embedding
(iv) CedarOil
 Due tominimal sclerification created by cedar oil,it is an
appropriatetransparent agent forfine andsoft tissues. Super
hardtissues (e.g. skin tissue) anddense fibrous tissue arealso
easier to besectioned after immersing in cedar oil. However, this
oil is not useful forother common tissue sections because ofits
high concentration andweak penetrability.
 In recent years,a new type ofenvironment-friendly transparent
agent has appeared in market. Instead ofaromaticcompound,
the main components of thisnew reagent is alkanes, andit can
be used to replace xylene.

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(D)Immersion
 After transparentizing, the tissue can be immersed in molten
paraffin waxso that it adsorbs the wax-substituting transparent
agent. Based upon the melting point ofwax,immersion should
be performed at 54-64°C.
Paraffin Embedding
(E)Embedding
 This is a process oftreating the tissue in a paraffinbox so that the
paraffin waxcools down andsolidifies. The treatment conditions
(using ethanol andxylene as an example) areshown in the table
below. After cooling is completed, the tissue will be ready for
sectioning andsuitable for storage.

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Paraffin Embedding
Paraffin Embedding Treatment Conditions
Step Reagent
Time (Hours
)
1 75% Ethanol 0.5 to 2
2 85%Ethanol 0.5 to 2
3 95%Ethanol. 2
4 95%Ethanol 2
5 95%Ethanol 2
9 Xylene 0.25
10 Xylene 0.25
11 Xylene 0.25
12 Paraffin Wax 0.5
13 Paraffin Wax 1 to 2
14 Paraffin Wax 1 to 2

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(A) Inactivation
 When either the horseradish peroxidase (HRP) oralkaline-
phosphatase (AP) system is applied for IHC,activation of
endogenous enzymes should be blocked orinhibited toavoid
producing non-specific binding.
(I) Endogenous HRP Inactivation
 Incubate the paraffin embedded section in 3% H2O2 for10 min
 Incubate the frozen section orcell section in solution composed
ofmethanol and3% H2O2 (v/v: 4:1) for30 min
Inactivation and Blocking
(ii) EndogenousAPInactivation
 Incubate the sample section in 0.1 mM Levamisole
Note: Levamisole cannot inhibit the AP activation ofendogenous
intestine tissue

 Residual sites on the tissue section may bindtosecondary antibody and produce follow-up false positive results. Therefore, serum from the
same species as the secondary antibody is commonly used forblocking. Animal’s autoantibody in the serum can bindtothe sites in advance.
Blocking should be done at room temperature for10-30 min (avoid excessive blocking).
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Inactivation and Blocking
(B) Blocking

 Formaldehyde fixation usually generates methylene bridges which cross-link proteins andtherefore mask the epitope ofinterest. It is
essential tounmask the antibody epitopes in ordertoallow the antibodies tobind,either by heat (Heat Induced Epitope Retrieval: HIER) or
enzymatic digestion (Proteolytic Induced Epitope Retrieval: PIER). To findthe optimal antigen recovery method, we suggest that you test both
HIER andPIERmethods, compare their results and optimize the method as needed.
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Antigen retrieval

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A)HIER
The HIER method canbeimplemented bymicrowave,highpressureor
waterbath. It breaksthemethylene bridgesandexposesthe epitopeto
allowthe antibodiestobindby continuouslyheating.ComparingtoPIER,
HIER hasa gentlerexperimental conditioninwhichusershavemorecontrol
overtheexperimentalparameters.However,the pHandbuffersforHIER
mustbe optimized. Thefollowing antigenretrievalreagentisrequired:
 0.01 Mcitratebuffersolution(pH 6.0)
 0.01 MPBSbuffer (pH7.0)
 0.05 MEDTA (pH 8.0)
 0.05 MTris-EDTA(pH 9.0)
 0.05 MTris-HCl(pH 1~12)
Antigen retrieval
(i) Microwave Method
 Place the sample section intoa microwaveable vessel where
antigen retrieval reagent is present
 Place the vessel inside a microwave oven
 Apply microwave radiation tothe sample for5-20 min

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(ii) High Pressure Method
 Place the sample section intoan appropriatevessel where
 Place the vessel inside a pressure cooker
 Turn on the cooker andheat the sample until it boils
 Once boiling starts, turn offthe cooker after the sample is
allowed toreach full pressure for1-4 min
Antigen Retrieval
(iii) WaterBath Method
 Place the sample section intoan appropriatevessel where
 Place the vessel andthermometer inside a water bath chamber
 Heat the sample to 92°Cin the chamber
 Remove the sample from the chamber after it is heated at 92°C
for20-40 min

Notes on HIER;
 The temperature andtime should be properly controlled for the antigen retrieval methods described above.
 To avoid originalprotein structure restoring, donot cool the sample section by taking it out of the buffer solution.
 The higher the temperature, the shorter the heating time (vice versa).
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Antigen retrieval

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(B) PIER
 Epitope can be exposed by incubation with proteases which can break the methylene bridges. The choice for digestion enzymes depends on
the antigenic components. Pepsin and bromelin areused for retrieving antigens in intercellular substance. Other enzymes can be used for
intracellular antigen exposure. PIERis suitable forretrieving moredifficult epitopes whilethe pHfor incubation is usually known. However,
PIERis aharsher method andcan damage tissue morphology.
Antigen Retrieval

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Antigen Retrieval
Enzyme Working Concentration Digestion Condition
Trypsin 0.05% to0.1% 37° C (10 to40 min)
Proteinase K 20 μg/ mL 37° C (20 min)
Pepsin 0.4% 37° C (30 to180 min)
Digestion Enzymes for PIER
Note: The reaction time can be increased for certain worn out tissue. Fresh trypsin solution
should be prepared with pH adjusted to 7.6 and used at 37 degrees Celsius.

IHC detection methods varyandarebased on the nature ofanalyze reporting and binding chemistry, among other factors. Three methods are
described here:
 Immunofluorescence (IF),
 Enzymatic and,
 Affinity.
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Detection

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(A)ImmunofluorescenceMethod
 Coons andco-workers developed the IF technique in 1941. This
technique is used for the rapididentification ofan antigen by
exposing it toknown antibodies labeled with the fluorescent dye
(i.e., fluorochrome) which produces light when excited by alaser
(e.g. argon-ion laser). Specific antibody binding can be
determined by the production ofcharacteristic visible light and
detected by afluorescence microscope. Tables 1 and 2 show
some ofthe common fluorechromes andtheir corresponding
excitation (λex) andemission wavelengths (λem) fornuclear
staining andIF, respectively.
Detection
(I) Principle
The indirect staining process involves three steps
 Primaryantibody binds specifically totargetantigen
 Secondary antibody labeled with fluorophore binds toprimary
antibody
 Fluorophore is detected via microscopy

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(ii) Tips: OperationsofFluorescence Microscope
 Operatethe microscopeaccordingtothe manual
 Turnonthe mercurylamp for5-15 mintostabilize thelight source
beforeuse
 Wearprotectiveglasseswhenadjustinglightsourcetoavoidharmful
ultravioletraystoeyes
 Intensityofhighpressuremercurylampwill dropifthe lamp isused
formorethan 90 min(Typically,thelamp iscontinuouslyusedfor1-2
hours)
 Photo-bleachingoccursifthe sampleisilluminatedby highpressure
mercurylamp formorethan 3 min(Note: Thesampleisgenerally
observedwithinone hourafterfluorescencestaining)
Detection
 Observe the samples intensively tosave time as light source is
limited
 Re-start the light source after turning it offfor 30 min orlonger
 Avoid using the light source several times during the day
 Observe samples immediately after staining
 There arefour levels forfluorescence intensity:
－:Non orweakly visible autofluorescence
＋:Clearlyvisible fluorescence
＋＋:Brightly visible fluorescence
＋＋＋:Dazzlingvisible fluorescence

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(iii) CounterstainingandStainedSampleStorage
(a) Nucleus Counterstaining
 Afterfluorescencestaining,counterstainshouldbe carriedout to
make morphologicalstructureofcellsand tissueswelldefinedand
specificfluorescencemoreeasilyvisible.Someofthe counterstaining
fluorochromesare:
 DAPI: classicbluecounterstainwhichis usedextensivelyfornucleus
and chromosomestaining(DAPI bindsselectivelytodsDNAwithout
backgroundstainingincytoplasm;DAPI hassemi-permeabilityto
livingcellsandcanbe usedtostainfixed cellsand/ortissuesections)
 Hoechst33342: primarycounterstainwhichisusedagainstyellow
fluorescence
Detection
(b) StainedSampleStorage
 After staining, the samples should be observed and imaged
immediately under a fluorescence microscope. They should be
mounted in buffered glycerol medium andstored at 4°Cforless
than one week if the image is not taken immediately. If anti-
fluorescence decay medium is applied tothe sample,
fluorescence signal may not decay significantly within one
month.

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B) EnzymaticMethod
 The enzymatic IHC technique was introduced by Nakane and
Pierce in 1967. Itidentifies antigens ofinterest by exploiting the
principle ofantibodies binding specifically toantigens. An
enzyme label is reacted with a substrate toyield an intensely
colored product that can be analyzed. The enzymatic technique
was developed with a similar principle tothe IF technique but the
two aredifferent as an enzyme is used tolabel the antibody for
the enzymatic method. The advantages ofenzymatic IHC over IF
IHC are;
Dectetion
 Fluorescence microscope is not required
 Accurate antigen location is enabled with better contrast ratio
 Stained samples can be stored fora long time
 Haematoxylin can beused as counterstain which enhances study
oftissue morphology
 End-product color can be easily identified andobserved by light
microscope (andalsoby electron microscopy due tohigh
electron density)
 Double andmultiple stains can be implemented

 For this method, the antibody used forantigen detection has been labeled with the enzyme before the reaction. After reacting with the
targeted antigen, the labeled antigen forms an antigen-antibody complex where the enzyme catalyzes a substrate toyield aninsoluble
colored product. Subsequently, the product can be analyzedby a light microscope orelectron microscope. The labeled-enzyme approach can
be done by direct orindirect detections.
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Detection
Labelled-Enzyme Antibody

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Types of detection method
Detection

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Direct Detection
 The direct method is a one-step staining method which involves
a labeled antibody (e.g. HRP-conjugated antibody) reacting
directly with the antigen of interest. The antigen-antibody-HRP
complex is then allowedto reactwith aDAB substrate for
staining.
 While the direct method is simple, rapidandhighly-specific, it
has low sensitivity andalimited range of primaryantibodies that
aredirectly labeled. Despite the shortcomings, the direct method
is commonly applied toscreen monoclonal antibodies before the
large-scale manufacturing process.
Detection
Indirect Detection
 The indirect method is a two-step process which involves an
unlabeled primaryantibody (first layer) that binds tothe target
antigen in the sample andan enzyme-labeled secondary
antibody (second layer) that reacts with the primaryantibody.
The secondary antibody must be raised against the IgG ofthe
animal species in which the primaryantibody has been raised.
For instance, if the primaryantibody is rabbit anti-human IgG, the
enzyme labeled secondary antibody could be goat anti-rabbit
IgG.

 First ofall,only a relatively small number ofstandardconjugated (labeled) secondary antibody is needed tobe generated forthe indirect
method. For example, a labeled secondary antibody raised against rabbit IgG,which can be purchased "off the shelf," is useful with any
primaryantibody raised in rabbit.With the direct method, it wouldbe necessary tolabel each primaryantibody for every antigen of interest.
 Secondly, the indirect method has greater assay sensitivity. Thirdly,various kinds ofcontrols could be designed andapplied with indirect
detection.
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Detection
Advantages of Indirect detection over Direct detection

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(ii) Unlabeled-EnzymeAntibody
(a) EnzymeBridgeMethod
Describedby apublicationfrom T.E. Masonand hiscolleaguesin1969, this
method isbasedonthe binding ofanenzyme labeltoatargetantigen
throughthe antigen-antibodyreactionsofan immunoglobulin-enzyme
bridgewhichconsistsofthe followingcomponentsinorder:
 Specificantiserumforthe tissueantigen(AnTAn)
 Antiserumagainstthe immune globulinofthe speciesfor AnTAn
 Specificantiserumpreparedagainstthe enzyme labelinthe same
speciesasAnTAn
 Enzyme label
Detection
(b)Peroxidase-Anti-Peroxidase (PAP) Method
 This method involves immunization ofarabbit/goat/rat antibody
with a HRP moiety toproduce an anti-HRP rabbit/goat/rat
antibody which would then bindtoanother HRP moiety to form
a stable polygon. The PAP approach excels due toits high
sensitivity andlow background fortissue staining.

 The IHC sensitivity can be improvedby employing a higher number of enzyme molecules boundtothe tissue. In this regard,the multiple
binding sites between the avidin andbiotinylated antibodies have been exploited for IHC signal amplification. Avidin, an egg white protein,
has four binding sites forthe low-molecular-weight vitamin biotin toform a large lattice-like complex. Beside avidin, there areother methods
which involve streptavidin which is a tetrameric biotin-binding protein that is isolated from Streptomyces avidin. The avidin andstreptavidin
methods workalmost identically as their structures arevery similar (they have very little amino acid homology). Avidin-Biotin Peroxidase
Complex (ABC) andLabeled Streptavidin Binding (LSB) arethe two most widely used affinity methods for amplifying the target antigen
signal.
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Detection
(C)AffinityMethod

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1. ABCDetection Method
Detection
 The methodinvolvesfoursequentialsteps
 Incubationofprimaryantibodywithtissuesampletoallowbinding to
targetantigen
 Incubationofbiotinylatedsecondaryantibody(whichhasspecificity
againstprimaryantibody) withtissuesampletoallowbinding to
primaryantibody
 Pre-incubationofbiotinylated enzyme(HRP orAP) withfreeavidinto
formlargeABC complexes(Biotinylatedenzyme and avidinaremixed
togetherina pre-determinedratiotopreventavidinsaturation)
 Incubationofthe abovepre-incubatedsolutiontotissuesample

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2. LSBDetection Method
Detection
 Thismethod usesan enzyme-labeledstreptavidintodetectthe bound
biotinylatedprimaryantibodyonthe tissuesection.It canalsobeapplied
ifthecomplex inthe ABC method istoo bigfor tissuepenetration.Dueto
itssmallersize,the enzyme-labeledstreptavidinisusedtoenabletissue
penetration.The LSBmethod canbe employedtoreplacetheABC
method forthe former’sabilitytoimprovesensitivityandreducesignal
further.The informationbelowdescribesthe generalstainingprocedure.
 Incubationofprimaryantibodywithtissuesampletoallowbinding to
targetantigen
 Incubationofbiotinylatedsecondaryantibody(whichhasspecificity
againstprimaryantibody) withtissuesampletoallowbinding toprimary
antibody
 Incubationofstreptavidin-enzymeconjugatetotissuesample

7. CHROMOGENS,COUNTERSTAINS
AND MOUNTINGMEDIA

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(A) Chromogensfor HRP
(I) DAB
 DAB (3,3’-Diaminobenzidine) is typically used as a signal
enhancer in conjunction with the HRP-based immunostaining
systems. The darkbrownend-product derived fromDAB is
insoluble in water andalcohol, stable andsuitable for long-term
storage. In addition, the end-product could be observed under a
light microscope orprocessed with OsO4 for observation under
electron microscopy. Hematoxylin, methyl green andmethyl
blue arethe compatible counterstains. Since DAB may cause skin
andbladder cancers, it is advised that personal protective
equipment should be used andskin/mucosa should be avoided.
Chromogens, Counterstains and Mounting Media
(ii) AEC
 After staining with AEC(3-Amino-9-Ethylcarbazole), the positive
area on tissue section changes todarkred. The end-product
derived from AECis soluble in organicsolvent andcannot be
stored on a long-term basis. SimilartoDAB, haematoxylin,
methyl green andmethyl blue aresome of the suitable
counterstains forAEC. Glycerin gelatin should be used as the AEC
mounting medium.

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(B) Chromogensfor AP
(i) BCIP/NBT
 Used in conjunction, BCIP(5-Bromo-4-Chloro-3-Indolyl-
Phosphate)/NBT (Nitro Blue Tetrazolium) is a widely accepted
chromogenic substrate used in the AP-based immunostaining
systems. After exposing toAP, the substrate changes tobluish
violet orblack violet. The end-product derived from BCIP/NBT is
insoluble in alcohol. Nuclear fast redandbrilliant green arethe
suitable counterstains forBCIP/NBT.
(ii) Fast RedTRSalt
 Fast Red is also used forthe colorimetric detection ofAP. Itsend-
product has a rose color and is soluble in alcohol. These
counterstains areused forthe Fast Red chromogens: methyl
green, brilliantgreen andsoluble hematoxylin.

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C)Counterstains
 Afterstainingthe targetantigenbyIHC, asecondarystainisusually
appliedtoprovidecontrastthat helpsthe primarystainmoredistinct.
While manyofthesestainsshowspecificityfordiscreteantigensor
cellularcompartments,otherstainswill deliverthestainingofa whole
cell.Someofthe mostcommoncounterstainsaredescribedas
follows:
 (i) Haematoxylin
 Haematoxylin,a naturaldye whichisextractedfromthe heartwood
ofthe logwoodtree,isusedfor cellnucleusstaining.Differentiation
refersto the processofusingreagents(e.g. 1% hydrochloricacidHCl
and alcohol)toremovethe colorcausedbyoverstainingornon-
specificstainingonsampletissues
 After running nucleolar staining (in aluminum haematoxylin)
anddifferentiation (in HCl andalcohol), the tissue section is
transferred from anacid solution toan alkaline solution (e.g.
ammonia water anddisodium hydrogen phosphate solution).
Duringthis process, the section will change from redbrown into
blue. This procedure is known as bluing.

52
HaematoxylinCategories
 (a) Mayer’s Haematoxylin is reddish violet andis valued for
several properties: low staining time, no perception andmetal
membrane as well as nopost-staining differentiation.
 (b)Harris Haematoxylin is purple red, widely used in H&E
staining andhas these advantages: fast staining, brightcolor,
clear nucleolar stains andwell defined tissue morphology.
Although metallic oxide may float on the Harris haematoxylin
solution after along period oftime, filter is unnecessary before
use as no perception will appear. Differentiation andbluing
should be carried out after staining with Harris Haematoxylin.
ii) MethylGreen
 Methyl green consists of metallic green microcrystals orbright
green powders. It becomes bluish green when dissolved in water.
This basic dye can be easily bounded with highly polymerized
DNA andchanges the nucleus togreen. Counterstain with
methyl green takes 2 to5 min which should be followed by
washing the sample, dehydration andmounting.
(iii) Nuclear Fast Red
 This counterstain will change the nucleus tored after applying to
the tissue section for2 to 5min.

53
(D)Mounting Media
 Amounting medium may beused toattach a coverslip or may
itself be used toreplace the coverslip. Generally, the medium
selection depends on a few factors including the chemical
compatibility with chromogen andcounterstain as well as the
preservation period.
(I) Neutral Mounting Medium
 It usually refers toan oily substance with pH 7.0 such as neutral
gum (resin). Before mounting, the sample should be treated with
dimethyl benzene, transparent anddehydrated for long-term
storage sections.
(ii) Water-Soluble MountingMedium
 Popularly used in IF staining forshort-term storage sections, this
mounting medium usually consists of50% glycerol.

54
Chromogens, Counterstains and Mounting Media forHRP andAP Enzymatic Systems
Enzyme Chromogen Counterstain Mounting Medium
HRP DAB Haematoxylin, Methyl Green, Methyl Blue Neutral
HRP AEC Haematoxylin, Methyl Blue Water Soluble
AP BCIP/NBT Nuclear Fast Red, Brilliant Green Neutral
AP Fast Red Haematoxylin, Methyl Green, Brilliant Green Water Soluble

56
(A)Positive Controls
 Every IHC experiment must include positive andnegative
controls. However, several other types ofcontrols can be
considered based on the type ofexperiment that you plan to
workon.
 Apositive control validates the staining ofyoursample and
confirms that the assay is working correctly. It will also help
optimize the conditions for future experiments. To identify
suitable positive controls, a goodstarting place is tocheck the
antibody datasheet any of these may be used as your positive
control.
CONTROLS
 You can alsocheck the Swiss-Prot orOmnigene database links
on yourantibody’s datasheet. The databases often include a list
of tissues that express your protein interest which can be used for
positive controls. In addition, the following resources arehelpful:
 GeneCards: It usually provides with relative levels ofexpression
in various tissues
 Human Protein Atlas: Itsprotein detection database has different
tissue types, cancers, andcell lines
 Pubmed: The literature search may giveyou ideas as towhich
tissues andcells express the protein ofinterest

57
(B) Negative Controls
 Negative controls, on the other hand, reveal non-specific binding
andfalse positive results. Obviously, negative controls should
not express your protein ofinterest. Acommon negative control
tissues areknock down (KD) orknock out (KO) tissue samples.
Controls
(C)No Primary Controls
 For this type ofcontrol, primaryantibody is not added tothe
sample. These controls areindicative ofnon-specific binding or
false positives that can result fromnon-specific binding ofthe
secondary antibody. Antibody dilution buffer with no antibody
added is incubated on the same sample under identical
conditions.

58
(D)Isotype Controls
 Isotype controls areantibodies ofthe same isotype (e.g. IgG2,
IgY), clonality, conjugate andhost species as the primary
antibody, targeting amolecule that is notpresent in your sample.
For example, the targetis generally achemical ora non-
mammalian protein. If used, incubate yoursamples with the
isotype control antibody ratherthan the specific primary
antibody (The concentrations andexperimental conditions
between the primaryandisotype control antibodies arethe
same).
Controls
E)EndogenousControl for Transfected CellLines
If yourexperiment involves testing ofrecombinant protein, it is
suggested that you include endogenous (non-transfected) positive
controls tovalidate yourresults andprovethat yourantibodies are
working. Detection of recombinant proteins presents a few challenges
that should be considered. First, the recombinant protein may fold
differently than the native form which could prevent the antibody from
accessing its targetepitope. This is especially common in cases with
tagged proteins. With this in mind,tags should always be placed on
either the N orC terminus ofthe recombinant protein. Secondly, it is
important tomake sure that the recombinant protein contains the
immunogen sequence for yourantibody.

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Immunohistochemistry (IHC) Principle

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