Seeing Is Believing: Use of Antibodies in Immunocytochemistry and In situ Hybridization

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Seeing Is Believing:Use of Antibodies in Immunocytochemistry

and In situ Hybridization

Glria Hman, PhDDepartment o Biology, Morgan State University

Baltimore, Maryland


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Sing Is Bliving: Us Antibdis in Immuncytchmistry andIn situ Hybridizatin

The Basics of Titrating AntibodiesThe goals o this chapter are to introduce newer

approaches or antibody use in immunocytochemistry(ICC) and nonradioactive in situ hybridization (nr-

ISH) and to oer help in understanding how chosen

approaches are optimized. We emphasize here inor-mation concerning which aspects o the techniquesare critical, and which are not, as well as insights intohow to troubleshoot these methods when they ail.

All the approaches are simply designed to link a mol-ecule in tissue (the antigen) to a visible or fuores-

cent product via an antibodys ability to serve as anintermediary between the antigen and the colored or

fuorescent product. The antigens may be the endog-enous molecules o the nervous system, injected trac-ers, transected gene products, or molecules attached

to probes or ISH in order to localize mRNAs. Allthese molecules have in common the trait o being

capable o generating antibodies when injected intoa host species; that is, they are antibody-generating

molecules, or antigens.

Although many companies now make antibodiesagainst a variety o antigens, knowing how to selectan appropriate technique or testing these antibodies

in tissue is less obvious. Use o kits or ICC visual-ization is not always successul, and it is not always

obvious who is at aultthe investigator preparing thetissue, the company selling the antibody, or the kit or

visualization. When unds are limited, being able toconvince a company that their reagents do not work

requires that one adequately test antibodies to estab-lish credibility. This section will ocus on the optimiza-tion o ICC staining protocols, stressing key variations

in conditions or conducting ICC tests that infuenceoutcomes. Details o how to perorm each o these

methods are published elsewhere (Homan et al.,1992; Berghorn et al., 1994; Homan et al., 2008).

The basic steps o ICC techniques are shown belowin simplied orm. Steps in parentheses are used in

some but not all protocols:

Fixationoftissue (Embedmentofthetissue) Sectioningoftissueblock

Permeabilizingofcellsforantibodyaccess Primaryantibodyapplication

Applicationofsecondaryantibodysolution Colordeposition

(Mountingofsectionsontoslides) Analysisofstainedmaterial

Hw shuld tissu b prpard?A eature that will infuence the range o concentra-tions that provides optimal antigen detection is how

the tissue is xed and processed. Most investigatorswill use standard buered ormaldehyde solutions

that are either purchased, or made in the lab romparaormaldehyde. While these are eective, or someantigens, xation will greatly infuence the outcome

(Homan et al., 1992). Two specic conditions areworth highlighting: I the antibody is generated by

conjugating the desired protein to a more antigenicmolecule, and the link is established by reaction with

carbodiimide reactions, then the tissue must be xedwith carbodiimide to expose the epitope that the

antibody recognizes. For conjugation that was madewith glutaraldehyde, a xative that contains eitherglutaraldehyde or another strong dialdehyde may

be required as well. Thus, knowing how the antigenwas prepared beore generating the antibody can be

important. There could be additional considerationsthat infuence selection o the xative. For example,

4% ormaldehyde solutions do not deactivate allenzymes, and tissue peroxidase is among the enzymesthat remain active. Thereore, i immunoperoxidase

methods are selected, measures to block endogenousperoxidase activity are required. I tissue is xed and

targeted or both ICC staining and ISH, whetherRNases are still active becomes important. Again,

enzyme activity is not eliminated by xation with 4%ormaldehyde but is totally eliminated when acrolein(2.5%) is added to the ormaldehyde solution.

How the tissue is processed ater xation can also be

a actor. Although counterintuitive, mounting sec-

tions onto slides beore ICC staining requires higherconcentrations o primary antibodies or optimalvisualization, despite the act that the sections areoten thinner than sections that are processed ree-

foating. Embedment o the tissue in paran mayextract the antigen rom the tissue, and thus, i sec-

tions mounted onto slides are used, xed rozen mate-rial may retain antigenicity better than tissue embed-

ded in paran. Use o sections mounted onto slidesregardless o whether they had been rst embeddedcan sometimes limit how easily the reagents gain

access to the tissue. This eature is especially notice-able when unxed rozen tissue is placed onto slides

and later xed. Staining or ICC is more dicultwith this tissue than would be seen i the tissue were

rst xed, and then cut and mounted onto the slides.As Dr. Toth will emphasize in Unraveling Compli-cations, later in this course, some tissue preparation

will mask the epitopes that allow antibodies to bind;in act, a variety o approaches have been devised or

restoring epitope availability.

In this investigators experience, maximal antigendetection is seen in xed, ree-foating sections; thus,this approach will be emphasized herein. When


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tissue samples are small or ragile, the tissue can beembedded in a solution o egg yolk and gelatin, xed

in 4% buered ormaldehyde, cut, and then success-ully processed while ree-foating. In using egg yolk

embedding, one must take care to eliminate contam-ination with egg white. Postembedment xation o

the blocks is needed and enables the egggel solutionto take on characteristics that resemble normal tissuein terms o resilience and stability.

It should also be emphasized that ree-foating sections

can be stored or protracted lengths o time (morethan 20 years) in antireeze solutions at 4C with no

loss o antigenicity (Watson et al., 1986; Homanand Le, 2004). This measure makes possible the lateruse o tissue with newly discovered tools without

requiring that additional tissue be generated.

Antibdy in hand: whr t bgin?One rst needs an understanding o how antibod-ies recognize antigens and the steps taken in ICC

methods or localizing antigens in the tissue. Typicalantibodies are o the IgG class, a cartoon o which is

shown in Figure 1. Note there are two domains thatconvey very dierent inormation: The Fc region(stem o the Y-shaped molecules) encodes inorma-

tion about the species o animal that generated theimmunoglobulin. Thus, the Fc region is constant

or all immunoglobulins the animal makes. Theregion o the Ig molecule that encodes or the

antigen is variable. It contains portions o the long

and short chains and is reerred to as the Fab domain.Two identical Fab regions bind to antigens.

Initially, immunocytochemical studies linked a fuo-

rescent molecule or enzyme directly to the antibody.This approach was only partly successul because

oten, the antigen binding sites became labeled andbegan blocking antigenantibody ormation. Most

methods used today are considered indirect inthat the molecule that is visualized or the enzymeis not linked to the antibodies specic or the anti-

gen (termedprimary antibodies) but more generally toantibodies generated against the Fc region, termedsecondary antibodies (Fig. 1).

Using antibdis r staining tissuUsing antibodies or staining tissue requires thatthe exact concentration or detection be used. Sys-

tematic dilution o the primary antibody determineswhere optimum staining can be obtained. The opti-mum concentration is infuenced by several actors:

the method used or detection (covered in the nextsection), the amount o antigen in the tissue, and the

antibodys anity or the antigen. Note that thereis no universal concentration that will always give

good staining or an untested antibody. O criticalimportance is that in todays world, antisera can be

eective at widely dierent concentrations: Someantibodies work well at a primary antibody concen-

tration o 1:1,000,000, whereas others give detectionat a concentration o 1:1000! Thereore, the most

eective test o antibody staining should include theollowing series:

1:1,0001:3,000

1:10,0001:30,000

1:100,0001:300,000

In other words, antisera should be tested with all otherreagents maintained at constant concentrations

over a 3-logarithmic (log) scale, in hal-log units.Our recommendation is to use the avidinbiotin

complex (ABC) method with nickel-enhanced di-aminobenzidine (NiDAB) as the chromogen. In thisway, variations in ading are not present, and one can

make use o some unique eatures that NiDAB oersto help the investigator to know whether the primary

antibody concentration is too high, or too low. Somesubsequent adjustment o the concentration between

those used or titration may be required ater examin-ing the tissue. Not only does the use o ABC methods

with NiDAB provide the most sensitive detection,but the color reaction (when used according to our

protocols) enables one to clearly distinguish betweenprimary antibodies that have a high degree o activ-ity but ail to stain because their concentration is too

high, and those that have no activity or stain weaklybecause their antiserum concentration is too low

(Homan et al., 2008).

Figur 1. Cartoon showing the structure o an IgG molecule andthe eatures important or use o immunoglobulins or ICC.


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One oten assumes that i staining is weak, thenthe primary antibody concentration is too low. This

assumption is not true. A eature poorly understoodby many investigators is that when too high a concen-tration o primary antibody is applied, the reaction is

quenched, and less product is actually produced. Thiseature is illustrated in Figure 2 with the titration o

an antibody against melanin-concentrating hormone(MCH). Note that at the highest concentration

depicted (1:3000), staining is minimal (and uneven).With dilution, however, relatively high backgroundstaining becomes more evident despite the appear-

ance o specic staining; as the optimum concentra-tion o primary antibody is approached, the back-

ground abates.

Note that NiDAB in titrations also shows a colorshit that is useul. When the serum concentrations

are too high, the positive staining (when present) hasa brown color despite the act that the NiDAB prod-

uct should be blue-black. Only when the primaryantibody is in the optimal range (1:100,000) does thestaining shit to blue-black. The lack o staining at

very high antibody concentrations appears as a resulto the inability o substrates to reach the enzyme.

For immunofuorescence detection, the quenchingo staining when primary antibody concentrations

are too high can also be observed; however, it can bemore dicult to distinguish low reactivity when theantibody concentrations are too high compared with

those produced by nonspecic staining when theantibody simply does not bind or is too dilute. The

exact concentrations that are too high vary witheach antibody preparation, and this is another reason

that titrations are so critical. With certain fuores-cence methods, eliminating background is dicult.

Th imprtanc cntrls: strngstaining is nt th sam as spcic

stainingIn the eld o ICC, debate continues over what con-stitutes an appropriate control or antibody reactions.

Too commonly, investigators use only the elimina-tion o the primary antibody. Eliminating the prima-

ry antibody rarely shows any reactivity unless highlevels o endogenous peroxidase activity are present:

It provides no inormation about antibody specicity,but only whether something (but not necessarily theIgs) in the antibody solution was required or stain-

ing. The goal o specicity controls is to validate thatthe tissue has the antigen, and that the antibodies are

revealing its correct location. The Journal o Compara-tive Neurology has required that controls or antibody

specicity include either use o a knockout mouse, orpresentation o Western blot data to validate stain-ing. While each is an excellent control, neither is

universally applicable. Some antisera that are able tostain tissue well in ICC reactions will not bind to the

antigen in a Western blot. Conversely, some antiserathat are useul or Western blots do not recognize the

xed ligand in tissue. Knockout controls are useulonly i the studies are conducted in mice (or the an-tigen is conserved across species) and the knockout

animal is available. Moreover, one has to be careulthat the knockout is a gene deletion or minimally,

deletion o the sequences encoding or the same por-tion o the protein ormerly contained in the antigen

used to generate the antibody.

False-positives can be dicult to interpret. In initialICC studies, the selection o preadsorption with theantigen was considered the best available way o vali-

dating antibody specicity. When a negative resultater antigen preadsorption is applied, the result sug-

gests that the antibodies are appropriately recogniz-ing the antigen. Positive staining ater preadsorption

can be used to identiy antibody populations to mol-ecules that may have contaminated the immunogen(or were purposely linked to it to increase immuno-

genicity); it can also identiy antibodies the host ani-

mal already had when it was immunized (Clayton andHoman, 1979). However, care must be taken: I thesame antigen used to make the antibody was contam-

inated with another protein, it will produce the alseimpression that the antibody is specic. One shoulduse the puried antigen rom another source i pos-

sible. Also, preadsorption controls do not eliminatestaining due to a similar, but not identical, antigen in

the tissue that possesses the same or closely relatedepitopes. For example, the antisera against leucine

enkephalin are not able to distinguish staining omethionine enkephalin present in nervous tissue.

Figur 2. Micrographs depicting a titration o an antibodyagainst melanin-concentrating hormone (MCH).


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In some circumstances, the antibodies are specic,but a simple preadsorption test ails. For example,

when the antigen has been modied by a chemi-cal reaction to link it to a larger protein to generate

the antibody, then a similar chemical modicationmay be necessary to enable antibodies to bind to the

antigen. To illustrate, or staining o histamine or sero-tonin in tissue, the nascent amine is not able to bindto the antibodies. Histamine antisera are generated

by reaction with carbodiimide; serotonin antibodiesare generated through reaction o the monoamine

with ormaldehyde. Thus, antisera against histaminewill be capable o recognizing only histamine that

was complexed to a protein in the presence o carbo-diimide, and serotonin must similarly be convertedto block the staining. Preadsorption should be con-

ducted with the same altered molecule.

eliminating backgrund stainingThere are many descriptions o additives that reducenonspecic binding o ICC reactants and thereby

lower background staining. Knowing a priori whetherany one will work is not always possible. There are

some exceptions. I protein conjugates were used ingenerating the antibody, there will be a need to addthe proteins used to increase antigenicity (thyro-

globulins, serum albumins, or limpet hemocyanin),though other additives may not be necessary. Most

requently, investigators add a 1% solution o nor-mal serum rom the species used or generating the

secondary antibodies. In general, we recommend

not adding anything unless the background stainingis high. An exception is when tissue is xed by

immersion or is poorly perused and has a great dealo residual blood. Red blood cells contain high levels

o peroxidase, and this activity needs to be blockedwhen employing ICC reactions that use peroxidase

reactions or detection. Red blood cells will alsopresent a problem or fuorescence i a red fuorophore

is selected, since hemoglobin fuoresces red. A ques-tion that is requently asked is how much eort shouldbe made to rescue poor staining. Procedures to reduce

background labeling can be eective, but i anantibody is poor or antigen detection, no amount o

treatment will make the procedure work well.

How the Methods Differ in SensitivityWhich mthd t us?A survey o the literature will reveal many methodsavailable or ICC studies. Which one to choose isoten based on the type o microscope available to the

investigator and the time needed to perorm the stud-ies. As will be apparent rom the remainder o this sec-

tion, dierences in the methods sensitivity (denedas the amount o product generated by the antigen)

will dictate a change in the optimum concentrationo the primary antibody. These dierences are pre-

dictable once a standard ABC/NiDAB titration hasbeen conducted. For the reasons cited in the previous

section, The Basics o Titrating Antibodies, I recom-mend using the ABC immunoperoxidase method with

NiDAB as the chromogen or titrating antibodies.

A nt abut mthds and thirnmnclaturThe most common indirect ICC methods used todayare classied as those that are immunofuorescent(link-

ing the antibody complex to fuorescent molecules)and those whose products are generated by enzymereactions and are visible under a light microscope.

The latter class uses either horseradish peroxidase oralkaline phosphatase reactions; a range o substrates

or each enzyme provides dierent colored products

in the visible range, some o which are also fuores-cent. Several newer methods comprise a hybrid othe two approaches and generate fuorescent prod-ucts through enzyme reactions, as will be discussed

in subsequent chapters herein by Drs. Roth and Toth(Tyramide Signal Amplication Strategies or Fluo-

rescence Labeling, and Unraveling Complications,respectively). The various methods are cartooned

below in ascending order o complexity/sensitivity.

Simple, indirect methods: primaryantibody + fuorophore/enzyme

conjugated to the secondary antibodyThis method is the least sensitive among com-monly applied immunofuorescence methods, yet is

the most commonly used (Fig. 3A). Only a limitednumber o molecules can be linked to the secondary

antibody. To ensure that sucient numbers o taggedmolecules bind in the antibody complex, a higher

Figur 3. Cartoons o the ICC staining using secondary anti-bodies that are either labeled with a uorophore (A), or labeled

with an enzyme (B) whose product ater reaction with a sub-

strate is colored.


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concentration o primary antibody is required thanwould be needed or more sensitive methods. The

enzyme-tagged approach provides greater sensitivitythan the fuorescent variant (Figs. 3B, 3C). A dier-ence can be noted between alkaline phosphatase and

peroxidase: For some alkaline phosphatase products,as the time o reaction is lengthened, more and more

product is ormed. With the most common substrate,nitro-blue tetrozolium, the cell can become so lled

with product that it loses compartmental distinc-tions. In contrast, the common peroxidase substratediaminobenzidine (reacted either in the presence or

absence o nickel salts) orms an insoluble precipi-tate product. This product will eventually cover the

enzyme and antigenic sites on the primary or secondaryantibodies, limiting the urther generation o product

while retaining the highest resolution. This eature iso great use in ISH reactions, as will be discussed sub-

sequently in Nonradioactive In situ Hybridization.

ABC: primary antibdy + bitinylatdscndary + strptavidin-furphr /strptavidin-nzymThis method oers greater numbers o molecules ordetection than are present in the previous approach,

since or each biotin molecule present on the sec-ondary IgG, our molecules o avidin can bind. As aresult, there is about a threeold increase in the

amount o fuorescent signal or each molecule obound primary antibody compared with that obtained

with the analogous method using a direct tagged sec-ondary antibody (Fig. 4). Translating this eature to

determine an optimal primary antibody concentra-tion, threeold less primary antibody is needed; and,since more signal is present, the amount o product

generated is greater. Thus, the time o exposure orfuorescence needed to photograph the specimen

is shorter, and or either fuorescence or enzymaticdetection, the signal-to-noise ratio is higher. The

enzymatic variant o this approach (Fig. 5), in whicha biotinylated enzyme is linked into the systemthrough an avidin link, is even more sensitive since

enzyme product urther amplies the signal. This

approach enables 50-100 less primary antibodythan the directly tagged secondary methods. ABCperoxidase is now the most commonly used o the

immunoperoxidase methods.

Tyramin signal amplicatin withbitinylatd tyramin as a substratThis method combines primary antibody + (bioti-nylated secondary + avidin peroxidase) and bioti-

nylated tyramine (with peroxide) as the substrate,ollowed by a streptavidin fuorophore/enzyme in-cubation (Fig. 6). It uses an increase in signal due

Figur 5. Cartoon o ABC ICC staining using an enzyme (E) link

in the complex.

Figur 6. Cartoon depicting amplifcation o the ABC complexthrough use o a tyramine signal amplifcation (TSA). Visualiza-

tion is accomplished with a streptavidin uorophore.

Figur 4. Cartoon o ICC staining with avidin-biotin complexes

(ABCs) using a uorescent avidin (streptavidin uorophore).


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to the generation o multiple biotin molecules atthe site o the antigen through an ABC enzymatic

reaction involving biotinylated tyramide (a substrateor peroxidase that, with peroxide, enables multiple

molecules o biotin to be deposited at the site othe antibody complex). Incubating the tissue with

streptavidin linked to a fuorophore completes thereaction. The result is a highly amplied fuorescentsignal (Berghorn et al., 1994). The fuorescent prod-

uct is bright, and the sensitivity o the overall reac-tion is ar greater than that produced by any o the

other fuorescence methods. Primary antibody con-centrations to obtain optimal product are 100-200

lower than would be needed or the directly tagged,secondary methods. As will be discussed in a subse-quent chapter (Tyramide Signal Amplication Strat-

egies or Fluorescence Labeling), the overall reac-tion can be urther stimulated by using buers with

high salt concentrations. It can be used with peroxidasediaminobenzidine (DAB) detection, but since

it becomes so sensitive, it is quite dicult to useowing to the crowding o complex components(as discussed in The Basics o Titrating Anti-

bodies, above).

Gnral rcmmndatinsFor all the methods, the incubation time with theprimary antibody is 24-48 hrs at 4C. Incubation

with secondary antibodies and subsequent reagentsare applied as detailed in our recommended protocols

(Homan et al., 2008).

The most important take-home message when

selecting a method or staining is that the concentra-tion o primary antibody needed to obtain optimal

localization is determined by the sensitivity o themethod. Thus, the same dilution o primary anti-

body does not result in optimal staining across meth-ods. Although one oten is given a recommended

antibody concentration or ICC by a company,unless the method used or screening is speci-ed in detail and includes the conditions used or

preparing the tissue and perorming the assay, thesuggested concentration can be orders o magnitude

dierent rom what would provide reliable stainingin your experiment.

Selecting Methods for Double-LabelingThis section will cover some o the basic strategiesor double-labeling, how to select the right combina-

tions o methods, and how each o the methods isperormed. Although the results presented here are

based on use o tissue that is processed ree-foating,with some adjustment they can be applied to any

tissue preparation. Using sections that are stainedwhile reely foating gives reagents optimal access to

the tissue and allows excess reagents to be eectivelyrinsed away, giving products or detection the high-

est sensitivity and signal-to-noise ratios.

Slcting th right cmbinatin mthdsBeore beginning to stain tissue or two dierentmolecules, one must evaluate each separately and

determine whether the two molecules are likely tobe ound in the same or dierent structures within

the tissue. The options or double-labeling are dier-ent i both molecules are within the same cells andin the same cellular compartment in approximately

the same amounts, rather than in dierent com-partments within the same cells or in two dierent

cells. Whether the two antibodies used to stain the

tissue are generated in the same or dierent speciesalso can infuence which methods can be combined.Beore outlining double-labeling strategies, it isimportant to recognize that each method or ICC

oers a number o advantages and disadvantages.

Combinations or double-labeling will use the ol-lowing approaches:

(1) The ABC approach detected with the per-oxidase substrate DAB, or DAB reacted in

the presence o nickel salts (NiDAB).

(2) The ABC approach detected with a strepta-vidin fuorophore.

(3) Immunofuorescence using biotinylated TSAwith detection via strepavidin linked to fuo-

rophores (Berghorn et al., 1994; Homan etal., 2008).

(4) Indirect staining with a secondary antibody

linked to alkaline phosphatase, ollowed byreaction with fuorescent substrates or theenzyme (McDonald et al., 2000).

(5) Indirect immunofuorescence using second-

ary antibodies prejoined to a fuorescentmolecule.

Cnsidratins which mthds tcmbinA decision o which methods will be best or

double-labeling requires understanding, most impor-tantly, where within the tissue the two antigens are

located. Ater examining each antigen, the choicesare as ollows.


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Antigens present in two dierent cells or two separatecellular compartments in the same cell

ABC peroxidase (NiDAB) + ABC (DAB)

ABC peroxidase (DAB or NiDAB) + anyfuorescence method

Alkaline phosphataselinked secondary

antibodies (fuorescence detection) + any otherfuorescence method

only when primaries are generated in dierent species

Direct-tagged secondary antibodies or both

only when primaries are generated in dierent species

TSA-amplied fuorescence + fuorescence-tagged secondary

ABC fuorescence + TSA amplied fuorescencetwo methods using biotin cannot be combined

Staining tissue to reveal the labeling o two antigens

present in dierent cells, or in distinctly dierentcompartments in the same cell, enables the greatest

fexibility or selecting a method. One could easilyuse two dierent immunoperoxidase reactions (i.e.,

NiDAB and DAB; Fig. 7); one immunoperoxidase

step and one o the immunofuorescence meth-ods (Fig. 8); or selected double-immunofuorescent

approaches (Figs 9, 10, and 11). In considering

approaches that use immunoperoxidase reactionswith DAB as a substrate (with or without nickelsalts), it is important to recognize that, as the oxi-dized substrate is deposited in the tissue, the DAB

product is insoluble and thus does not diuse romthe site o the enzyme. Eventually, the DAB product

will cover the sites where the enzyme is located andprevent any urther reaction rom occurring. Since

the enzyme lies over the antigenantibody com-plexes, these also will be masked by the precipitate.This eature limits the spread o product but, more

important, prevents urther reactions with any othe complex components. A second application o

secondary antibody will not bind to the reacted pri-mary antibody; thus, it is possible to use sequential

reactions with two ABC peroxidase methods even ifthe two primaries were generated in the same species.

A variant o this method uses one o the fuores-cent strategies along with ABC immunoperoxidase

labeling (Fig. 8). The ABC method is perormedrst, ollowed by the immunofuorescence method.

When NiDAB is used in this type o protocol, anyfuorescent molecule will be useul. I DAB rather

than NiDAB is used, fuorophores that emit in the

red wavelengths ~600 nm cannot be used, but those

that emit at lower wavelengths can.

I dual fuorescence methods are selected, the mostimportant consideration will be whether the two pri-mary antibodies were generated in the same or dier-

ent species. I primary antibodies were generated in

dierent species, then alkaline phosphatase-linkedsecondaries can also be used or one, and eitherbiotin-based immunofuorescence (TSA-amplied

or standard ABC) or direct fuorophore-tagged sec-ondary antibodies can be applied or the second

(Fig. 9). Two fuorescent-tagged secondary antibodiescan also be applied (Fig. 10). In addition, it is possi-ble to combine TSA amplication with fuorescence

using a fuorescent-tagged secondary (Fig. 11).

Which o these fuorescence methods will work bestdepends on a number o actors that include the ol-

Figur 7. Double-labeling strategy that uses two reactions o

peroxidase (P) or antigen detection; one with NiDAB as the sub-

strate (black) and the other with DAB as the substrate (brown).

Provided the NiDAB reaction is run to completion, the brown

and black stains do not mix.

Figur 8. Double-labeling that employs the ABC reaction orthe frst antigen ollowed by uorescence with a tagged second-

ary antibody or the second.


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lowing: (1) the amount o antigen present (i verydierent, then it is best to ampliy the least abundant

o the two antigens to better equalize their detec-tion) and (2) the relative volumes o each antigen in

the cell compartment. I one antigen occupies a verysmall subcompartment, it is best detected i TSA

amplication is applied.

Amplication may also be helpul i the signal-to-

noise ratio o one o the antigens is low while usingfuorescence with a direct-tagged secondary. Under

these conditions, amplication will better separatebackground staining rom specic labeling. Our rec-

ommendation is that TSA amplication be used orone o the two antigens unless both are present inhigh abundance.

I the two primary antisera are generated in the same

species, TSA amplication can be combined witha method that uses a directly conjugated secondary

antibody (Shindler and Roth, 1996). Biotin-basedapproaches in double immunofuorescence tech-niques can only be used once, however.

Antigens present in the same cells and in the same

cellular compartment

ABC peroxidase (NiDAB) + ABC (DAB)The two products are not easily distinguished in the

same compartment

ABC peroxidase + any fuorescence methodThe opacity o the DAB product obscures

fuorescence detection

TSA-amplied fuorescence + fuorescence-

tagged secondary

Alkaline phosphataselinked secondary

antibodies (fuorescence detection) + anyother fuorescence method

only when primaries are generated

in dierent species

Direct-tagged secondary antibodies or bothonly when primaries are generated in dierent

species and detection is strong

ABC fuorescence + TSA amplied fuorescenceAvidin-biotin interactions make separating

two products impossible

When two antigens are expected in the same cellular

compartment in approximately equal amounts, thechoices or staining strategies are more limited than

when the two antigens are ound in separate cells or

Figur 9. Double-labeling that uses alkaline phosphatasetagged secondary antibodies or one antigen, and uorophore-

linked secondary antibodies or the second.

Figur 10. Double-labeling using two uorophore-tagged

secondary antibodies.

Figur 11. TSA amplifcation o the product o the frst antigen/

antibody series ollowed by use o a direct uorophore-tagged

secondary or the second provides increased sensitivity o the

detection o the frst antigen.


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in very dierent compartments. Only fuorescencemethods can eectively distinguish the two anti-

gens within the same compartments. In using twofuorescent methods, one must choose a way toachieve sucient sensitivity rom the method in

order to see each antigen. Should one series oreactants be amplied? Are the two antibodies gen-

erated in the same species? Is one antigen muchmore abundant than the other? In addition, which

fuorophore should be used or which antigen needsto be considered. Can the microscope used discrimi-nate the two fuorophores?

Without very sophisticated equipment, eliminat-

ing the excitation range o the rst fuorophore thatoverlaps with that o the second fuorophore can pro-

duce alse-positives. False-positives can result i usingone antigen that is in much higher abundance than

another, or one that is amplied versus one that is not.This will take place i a green-fuorescent fuorophoreis applied to the reaction with the highest abundance

and coupled with a standard red fuorophore. To seewhy this occurs requires an understanding o the spec-

tral characteristics o each fuorescent dye (shown inFig. 12, or Alexa dyes 488 and 548two o the com-

monly used green and red fuorophores). The sameissue pertains to fuorescein and rhodamine, or orCy2 and Texas Red fuorophores, or or some o the

newer dyes such as DyLight molecules [ThermoScientic, Rockville, IL]).

When both fuorophores are present in equal amounts,the two colors remain separate. Very little green fuo-rophore is excited by the wavelengths that excite thered fuorescent molecule, despite the act that a slight

tail in the excitation spectrum is ound in the rangewhere red fuorescence is excited (Fig. 12A). I, how-

ever, the amount o the green fuorescent molecules isnow greatly increased over that o the red fuorescent

one, a considerable amount o green fuorophoreextends into the wavelengths that enable emission ofuorescence in the red range (shaded areas Fig. 12B).

As a result, with lters in place or visualizing the redfuorescence, the green fuorophore will appear red.

When it is the red fuorophore that is amplied (Fig.12C), no greater bleed through o the green fuoro-

phore is ound, keeping the fuorescence o the twomolecules optically separate. Thus, investigators areurged to use fuorescent molecules that emit in the

red wavelengths or amplied or highly abundantsignals. One can determine ahead o time whether

bleedthrough will be a problem. A critical controlstep beore proceeding to put two fuorescent mol-

ecules together is to take each reaction alone and tovalidate that the fuorescence can be generated onlyby the appropriate lter combination.

Succssul antign lcalizatin indubl-labling rquirsundrstanding that dirntmthds rquir dirnt primaryantibdy cncntratins t achivptimal stainingAs was mentioned in How the Methods Dier inSensitivity, above, there are dierences among thevarious methods with respect to how much primary

antibody is required or optimal labeling. I the choiceo primary antibody or double-labeling is not in the

most eective range, unexpected and conusingresults can be obtained.

To illustrate this point, we present the ollowingscenario. Two antisera are used in a study to deter-

mine the axon innervation pattern o transmitter

#1 onto neurons expressing transmitter #2. ABCimmunoperoxidase with NiDAB detection was usedor transmitter #1 in the axons. DAB staining with

an ABC reaction was used or transmitter #2 toreveal the neurons being innervated. Single-labeling

Figur 12.A, Absorbance (excitation) spectra and emissionspectra highlighting the patterns that would be seen using a

standard uorescence microscope. The boxed areas show the

bandwidth o common flters or red uorescence. Note that

there is very little absorbance rom the green uorophore with

use o the red flters. Thus, the green uorophore would not

result in substantial red signal.


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using each method generated acceptable results

(Figs. 13A, 13B), but or the one using NiDAB,the time o staining was shortened to avoid back-

ground staining. When the double-staining reaction

was perormed, the results were striking (Fig. 13C). No evidence o the second (brown) antigen was

Figur 12.B, I the amount o green uorophore generated in the reaction is greatly increased, then a substantial amount o

uorescence rom the green uorophore absorbs light in the red range (gray area on let). This would be seen as red uores-

cence (gray area on right) and alse-positive double-labeling results. The section had only been reacted with green uorophore,

yet it appears to be uorescing both green and red.

Figur 12.C, When the amplifcation reaction or immunouorescence is reversed (ampliying the red uorophore), no alse-positives are ound, since no appreciable absorbance/emission o the green uorophore in the range o the excitation/emission

flters is ound.


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ound, and the background in the tissue turned darkbrown. With this result, it was tempting to conclude

that something had gone wrong with the staining othe second antigen with DAB, when in act it wasthe staining o the rst antigen that caused all the

problems. When using an antibody at a concentra-tion that is too high but employing a staining time

that is too short to complete the reaction o depositedperoxidase, staining will nish upon reexposure to

DAB. Brown-black mixing will occur, and oten, noproduct rom the second antibody complex will beound. Diluting the rst primary antibody solves the

problem (Fig. 13D). Similar considerations must beapplied i fuorescence methods are used and antisera

concentrations are too high or optimal detection.This eature suggests that crowding o antibodies

or the complexes that label them is responsible orthe discoloration.

Nonradioactive In situHybridization: A New Twiston an Old ThemeThe characterization o the genomic codes orhumans, mice, rats, and a variety o other species has

advanced our understanding o the composition andunction o the nervous system. Techniques or mea-

suring levels o RNA using real-time (rt) PCR andquantitative rtPCR have made it possible to assesshow gene expression changes under physiological and

pathological conditions. This, too, has greatly addedto our knowledge o the nervous systems unction and

plasticity. Yet it must be appreciated that the nervoussystem is a highly diverse organ system. Eventually,

one must ask where detected genes are expressed, andwhether observed changes are seen in all, or only inselect, populations o cells. It is within this realm that

ISH strategies provide an eective means or deter-mining where genes are expressed.

Two basic approaches are used or ISH: isotopic

(radioactive) methods and nonisotopic (nonradio-active) approaches. Isotopic ISH methods employ

nucleotide sequences complementary to the cellsRNA template that have an incorporated radio-active signal (3H, 35S, 32P) on one o the nucleotides.

When reacted (hybridized) with the tissue, the labelis detected by exposing lm or emulsion to the sec-

tion in order to reveal the radioactive label. Mostquantitative assessments with ISH use this approach.

Figur 13. Proper antibody titrations are important or double labeling.A, Shortened staining times or the frst o two sequen-

tial antibody/antigen series may produce good staining. B, Verifcation o the second antigen/antibody staining series alone also

shows clear and high quality labeling. C, When combined, no clear double labeling is ound; high brown background is present.

D, When the frst primary is properly diluted, then clear double labeling is possible.


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One eature o isotopic ISH is that the detected signalis ound at some distance rom the labeled nucleotide.

As a result, isotopic methods oten lack precise cellu-lar resolution o the signal: Invariably, there is spread

o the signal as it travels rom the tissue to the emul-sion or lm. In addition, there can be some decay in

the strength o the signal (depending on the isotope)that worsens as a unction o the distance betweenthe labeled nucleotide and the emulsion. Thus, with

standard 20 m sections, only the part o the sectionclosest to the surace is accurately monitored.

Nonisotopic or nr-ISH avoids problems o signal

spread or decay by enabling the label to be detect-ed within the tissue at the site o the hybridizedsequence. Two labels are commonly used or light

microscopic nr-ISH: digoxigenin and biotin. Bothare small, planar molecules and are easily stained

using antibodies against these molecules. For digoxi-genin, antibodies that are either unlabeled, or are

conjugated to alkaline phosphatase, are commer-cially available. The latter have been employed inmost studies with digoxigenin. Use o digoxigenin in

nr-ISH labeling is presented in detail in the chap-ter In situ Hybridization Using Antibody-Based

Methods by Dr. Baskin.

It should be apparent that use o either digoxigeninor biotin-tagged nucleotides, ollowed by ICC

detection, can be viewed as a simple modicationo standard ICC; many o the same approaches that

are useul or ICC will be useul or ISH. Indeed, ourgroup and others have tested this hypothesis directlyater realizing that, just as ICC detection o molecules

is greatly improved by use o reely foating tissue, sois ISH.

Us digxignin-taggd nucltidsin r-fating sctins prcssdr ISHAs shown in Figure 14, digoxigenin-labeled nucle-

otides that are subsequently stained using alkalinephosphataselinked anti-digoxigenin and nitro-

tetrozolium blue as the substrate provide strong, clearstaining signals in tissue. Background labeling ismuch lower than is usually ound with isotopic meth-

ods. Details o the basic method have been published(Elias et al., 1998).

Prxidas-labld anti-digxigninA variant o the enzyme-linked nr-ISH employs per-

oxidase linked to the anti-digoxigenin. As Figure 15shows, this method also provides a clear signal o

the probe.

Fluorescein-labeled nucleotidesOne approach that is theoretically simple is the use

o a fuorescent molecule tagged to a nucleotide. Oneo these, fuorescein, is sometimes used or detectingDNA sequences in cells. For nr-ISH, our experience

indicates that no fuorescent signal can be obtainedrom the fuorescent tag, but when ICC is applied us-

ing an anti-fuorescein antibody, ollowed by ABCdetection o the antibody complex, a clear signal can

be obtained (Fig. 16).

Bitin-labld nucltids r ISHMany years ago, scientists stood up at a meeting anddeclared that nr-ISH with biotin-labeled nucleotideswas associated with too high a level o background

Figur 14. Cartoon o nr-ISH that uses digoxigenin-tagged nu

cleotides (black triangle) and antibodies against digoxigenin tha

are conjugated to alkaline phosphatase (AP). The most common

substrate or the enzyme reaction is nitrotetrozolium blue (NTB)

and an example is shown in the micrograph insert.

Figur 15. Variant o the nr-ISH with digoxigenin-labeled nucleotides that use anti-digoxigenin antibodies conjugated with

peroxidase (P). NiDAB staining o the peroxidase is shown in the

micrograph insert.


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labeling, low signals, and was unable to be used reli-ably. O course, that statement has not been proven,

and may have been the result o a lack o understand-ing o how ICC should be optimized. For biotin-labeled nucleotides in ISH protocols, a wide range

o approaches can be used or detection. These cantake many orms: They can be simple (as would be

obtained with a streptavidin-fuorophore); they canemploy the linking o biotin to either avidin-perox-idase or avidin alkaline phosphatase; or they can be

made more complex through use o anti-biotin ol-lowed by ABC methods (Fig. 17) or ABC+TSA am-

plication (Fig. 18).

Our laboratory has developed and tested biotin-based

ISH approaches (Homan et al., 1995; Berghorn etal., 2001; Homan and Le, 2004; Koban et al., 2006;

Smith et al., 2006; Koban et al., 2008). The remain-der o this chapter will illustrate methods or detect-

ing biotinylated nucleotides. For this approach, someo the same principles outlined or ICC apply to ISH.Key among them is that the use o antisera to local-

ize the label requires that the antibody be properlytitrated. We have learned that i antisera against the

tags on the nucleotides are used under optimal condi-tions, reliable quantitative data are obtained. With

use o reely foating xed tissue, some steps requiredor slide-mounted tissue become unnecessaryin

particular, prehybridization or blocking steps. Instead,one need only perorm the ollowing simple steps:

Prepareprobe

Preparetissue.

Hybridizewithprobe(temperaturesmayneedto be reduced to about ~50C so that tissue isnot cookedknow that your body hybridizes

nucleotides at 37C)

Posthybridizationwashes

Developlabel

(ICCstartshere)

Tissue preparation

We have discovered that tissue that is xed with 4%paraormaldehyde:2.5% acrolein and then stored in

antireeze cryoprotectant well maintains both theantigenic proteins and mRNA (Homan and Le,2004). The critical eature o the latter is that acro-

lein appears to destroy the activity o ribonucleases(RNases) in the tissue. Acrolein xation also makes

the thin sections (25 m) more resilient.

Probe selectionOur experience indicates that cDNA or riboprobesthat are between 350 and 800 bases in length are

optimal or generating enough signal to detect, and

Figur 17. Nucleotides conjugated to biotin enable ABC peroxi-

dase detection with anti-biotin antibodies. Either DAB or NiDAB

is useul or the enzymatic reactions.

Figur 18. TSA amplifcation o the ABC reactions enables

increased uorescence signals or the ni-ISH reaction

(Cy3-strepavidin was used or the uorophore).

Figur 16. Nucleotides conjugated to uorescein (green tri-angles) may not provide sufcient uorescence alone, but anti-

uorescein antibodies enable ICC detection.


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or enabling good probe penetration into the tis-sue. Earlier studies by Trembleau and Bloom (1995)

applied a similar approach by using biotinylatedoligonucleotide detection and examination at both

the light- and electron-microscope level. Their datashowed that the cytoplasmic mRNA clusters accu-

rately refected expected compartmentalization othe mRNA, with localization at the endoplasmicreticulum, though not the mitochondria or Golgi ap-

paratus. Probe sizes are optimal at 300-800 bp. Very-low-abundant sequences may be dicult to detect,

and or those, radioactive ISH may be the best tool.

HybridizationIn our experience, biotinylated UTP works bestor nr-ISH. Working with multiple probes used or

biotin-based ISH indicated that concentrations othe probe in the range o 600-800 ng/kb/ml provide

reliable mRNA detection. A range o temperaturesor hybridization rom 37C to 60C revealed that

a maximum temperature o 50C maintained probespecicity without damaging the foating sections.Protein antigenicity is also well maintained through

this procedure. Following rinsing and treatment withbuers to remove unreacted probe, the tissue is ready

to begin ICC detection.

Probe detectionIn theory, detecting the label on the probe is no di-

erent or ISH than or ICC. Our standard strategy isto obtain an antibody against the label, titrate it as

we recommended or ICC, and then use the optimalconcentration or batch staining. As with other ICCreactions, incubation with the primary antibody lasts

48 hrs at 4C. ICC o biotin in ISH using ABC per-oxidase staining has some key dierences rom the

routine ICC method, since the level o backgroundstaining has to be kept extremely minimal in orderto see the ne, small RNA clusters in the tissue. The

secondary antibody concentration is lowered, theABC reactant concentrations are lowered, and the

NiDAB/H2O2 concentrations are reduced as well.Titration o the antibodies in nr-ISH is important,

whether digoxigenin or biotin tags are used (Fig. 19).

When anti-digoxigenin is too concentrated, back-ground labeling is increased.

Quantifcation o nr-ISH signals

Enzymatic detection o immunoreactive products canbe useul or determining the level o RNA present in

tissue. For ABC methods using peroxidase detection,this eature is reinorced by the act that the productmasks the enzyme sites, limiting urther generation

o product. As a result, the amount o product willaccurately refect the amount o incorporated label.

This eature is shown in Figure 20, which depicts

vasopressin mRNA under conditions where the geneis expressed normally or suppressed ater the animalsbecome hyponatremic (Homan et al., 1995). The

ISH signal clearly refects the level o mRNA, andquantication o the nr-mRNA clusters provides a

reliable estimation o mRNA expression (Berghornet al., 2001; Koban et al., 2006; Koban et al., 2008).

ReferencesBerghorn KA, Bonnett JH, Homan GE (1994) cFos

immunoreactivity is enhanced with biotin ampli-

cation. J Histochem Cytochem 42:1635-1642.

Berghorn KA, Le WW, Sherman TG, Homan GE

(2001) Suckling stimulus suppresses messengerRNA or tyrosine hydroxylase in arcuate neurons

during lactation. J Comp Neurol 438:423-432.

Figur 19. As with any o the ICC methods, a proper titratio

o the anti-tag antibody (shown or anti-uorescein) is require

to optimize the assay.

Figur 20. Nonradioactive ISH using biotinylated riboprobe

enables useul and accurate quantitative assessment o mRNA

levels. A) a section through the hypothalamic paraventricula

nucleus rom a control rat reacted or vasopressin mRNA. B) Sec

tion through the same region rom a rat that was hyponatremic

or 7 days shows markedly suppressed mRNA expression.


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Clayton CJ, Homan GE (1979) Immunocytochemi-cal evidence or anti-LHRH and anti-ACTH activ-

ity in the F antiserum. Am J Anat 155:139-145.

Elias CF, Saper CB, Maratos-Flier E, Tritos NA, Lee

C, Kelly J, Tatro JB, Homan GE, Ollmann MM,

Barsh GS, Sakurai T, Yanagisawa M, Elmquist JK(1998) Chemically dened projections linking the

mediobasal hypothalamus and the lateral hypotha-lamic area. J Comp Neurol 402:442-459.

Homan GE, Smith MS, Fitzsimmons MD (1992).Detecting steroidal eects on immediate early

gene expression in the hypothalamus. Neuropro-tocols 1:52-66.

Homan G, Berghorn K, Knapp L, Le W, Sherman T(1995) Physiological stimulation o vasopressin

and oxytocin neurons: perspectives rom Fosactivation. In: Neurohypophysis: Recent prog-

ress o vasopressin and oxytocin research (SaitoT, Kurokawa K, Yoshida S, eds), pp 151-164.Amsterdam: Elsevier Science.

Homan GE, Le WW (2004) Just cool it! Cryopro-tectant anti-reeze in immunocytochemistry and

in situ hybridization. Peptides 25:425-431.

Homan GE, Le WW, Sita LV (2008) The

importance o titrating antibodies or immunocyto-chemical methods. Curr Protoc Neurosci 2.12

(in press).

Koban M, Le WW, Homan GE (2006) Changes in

hypothalamic corticotropin-releasing hormone,neuropeptide Y, and proopiomelanocortin gene

expression during chronic rapid eye movement sleepdeprivation o rats. Endocrinology 147:421-431.

Koban M, Sita LV, Le WW, Homan GE (2008)Sleep deprivation o rats: the hyperphagic response

is real. Sleep 31:927-933.

McDonald TJ, Le WW, Homan GE (2000) Brain-

stem catecholaminergic neurons activated byhypoxemia express GR and are coordinatelyactivated with etal sheep hypothalamic para-

ventricular CRH neurons. Brain Res 885:70-78.

Shindler KS, Roth KA (1996) Double immunofuo-rescent staining using two unconjugated primaryantisera raised in the same species. J Histochem

Cytochem 44:1331-1335.

Smith JT, Popa SM, Cliton DK, Homan GE,

Steiner RA (2006). Kiss1 neurons in the ore-brain as central processors or generating the preo-

vulatory luteinizing hormone surge. J Neurosci26:6687-6694.

Trembleau A, Bloom FE (1995) Enhanced sensitivityor light and electron microscopic in situ hybrid-

ization with multiple simultaneous non-radioac-tive oligodeoxynucleotide probes. J HistochemCytochem 43:829-841.

Watson RE, Wiegand SJ, Clough RW, Homan GE(1986). Use o cryoprotectant to maintain long-

term peptide immunoreactivity and tissue mor-phology. Peptides 7:155-159.

Documents

Seeing Is Believing: Use of Antibodies in Immunocytochemistry and In situ Hybridization