Multiple Labelling Using Fluorescent Labelled Secondary Antibodies

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Multiple Labelling Using Fluorescent Labelled
Secondary Antibodies
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Simultaneous detection of more than one antigen
depends on at least two important criteria

• Secondary antibodies that (a) are derived from
  the same host species so that they do not
  recognise one another, (b) do not recognise
  other primary antibodies used in the assay
  system, (c) do not recognise immunoglobulins
  from other species possibly present in the assay
  system, and (d) do not cross-react with the
  tissues or cells under investigation.

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Simultaneous detection of more than one antigen
depends on at least two important criteria

• Probes that are well resolved (enzyme-reaction
  products, fluorophores, or electron-dense
  particles).

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Affinity-purified antibodies can be specifically
prepared to meet these criteria
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Selecting secondary antibodies

• F(ab')2 fragments are used when you wish to avoid
  binding of whole molecule, 2o antibodies to Fc
  receptors on cell surfaces.
• Alternatively, you can block the Fc receptors by
  incubating cells at 4C in a buffer containing
  sodium azide and normal serum from the host
  species of the labelled secondary antibody.
• However, if a primary antibody is not an F(ab')2
  fragment, it may also bind to Fc receptors, and
  blocking with normal serum from the host species
  of the secondary antibody may not always work.
• Caution Never block with normal serum or IgG
  from the host species of the primary antibody
  when using a labelled, secondary antibody.

• Whole Molecules or F(ab')2 Fragments?

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Selecting a secondary antibody

• Avoid the use of antibodies that have been
  adsorbed against closely related species
• Such antibodies may not react well with all
  subclasses of IgG, especially those subclasses
  which are most closely homologous to the species
  they were adsorbed against.
• E.g., do not use an anti-mouse IgG that has been
  adsorbed against rat IgG unless you are trying to
  detect a mouse primary antibody in rat tissue
  that contains rat immunoglobulin, or in some
  other tissue in the presence of a rat primary
  antibody.

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Secondary specificity

• Anti-IgG raised against whole IgG, reacts with
  both the heavy and light chains of the IgG
  molecule, i.e. it reacts with both the Fc and
  F(ab')2 portions of IgG. Anti-IgG also reacts
  with other immunoglobulin classes (IgM, IgA,
  etc.) since all immunoglobulins share the same
  light chains (either kappa or lambda).
• Anti-IgG, Fc fragment specific antibodies react
  with the Fc portion of the IgG heavy chain, and
  can be produced by adsorption against F(ab')2
  fragments. Sometimes, they are additionally
  adsorbed to minimise possible cross-reactivity to
  IgM and/or IgA. In such cases (anti-human,
  anti-mouse, and anti-rat), they are referred to
  as gamma chain specific.
• Anti-IgG, F(ab')2 fragment specific antibodies
  are produced by adsorbing against Fc fragments
  and therefore react only with the Fab portion of
  IgG. Since they react with light chains, they
  also react with other immunoglobulins sharing the
  same light chains.

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Secondary specificity (b)

• Warning Antibodies against one species may
  cross-react with a number of other species,
  unless they have been specifically adsorbed.
• Warning Bovine serum albumin (BSA) and dry milk
  may contain IgG which reacts with anti-bovine
  IgG, anti-goat IgG, anti-horse IgG, and
  anti-sheep IgG antibodies. Therefore use of BSA
  and/or dry milk to block or dilute these
  antibodies may significantly increase background
  staining and reduce antibody titre.

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Minimised cross reactivity

• Minimised cross reactivity antibodies will have
  been tested by ELISA and/or adsorbed against the
  IgG and serum proteins of other species.
• Recommended when the possible presence of
  immunoglobulins from other species may lead to
  interfering cross-reactivities.
• Use Caution when considering antibodies adsorbed
  against closely related species as they have
  greatly reduced epitope recognition and may
  recognise some monoclonal antibodies very weakly.
• Anti-Mouse IgG (min X Rat and other species) and
  Anti-Rat IgG (min X mouse IgG and other species)
  have diminished epitope recognition. Most
  multiple-labelling experiments require the use of
  minimised cross reactivity antibodies to minimise
  cross-reactivities to other species.

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Example of the use of minimum cross reactivity
secondaries
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Primary Antibodies from the Same Host Species -
Blocking and Double Labelling with Fab Fragments

• Fab fragments of affinity-purified, secondary
  antibodies are used to sterically cover the
  surface of immunoglobulins for
• Double labelling primary antibodies from the same
  host species,
• To block endogenous immunoglobulins on cell or
  tissue sections.

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Why monovalent Fab fragments?

• Monovalent Fab fragments of secondary antibodies
  may be used for these purposes for the following
  reasons
• Whole IgG molecules and F(ab')2 fragments of IgG
  have two antigen binding sites.
• After binding to its primary antibody (for
  example, goat anti-mouse IgG binding to the first
  mouse primary antibody), most of the secondary
  antibodies will still have one open binding site,
  which can capture the second primary antibody
  from the same species (for example a second mouse
  IgG primary antibody).
• Consequently, overlapping labelling of the two
  antigens will occur.

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More about Fab fragments

• It is not necessary to use Monovalent Fab
  secondary antibodies when primary antibodies from
  the same host species are different classes of
  immunoglobulins, such as IgG and IgM.
• It is also unnecessary to use these when primary
  antibodies from the same host species are
  different subclasses of IgG, such as Mouse IgG1
  and Mouse IgG2a. In these cases, class-specific
  or subclass-specific antibodies may be used to
  distinguish between the two primary antibodies.
• Remember that Fab fragments havent been adsorbed
  to remove cross-reactivities to other species,
  and they might contribute to some degree of
  background staining for certain applications.

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Possible protocols used for double labelling
using Fab fragments

• The success of these experimental designs is
  unpredictable and may require some empirical
  manipulations.
• Trying different concentrations of reagents in
  each step or switching the labelling sequence of
  the two antigens may sometimes influence the
  outcome.
• Blocking with an appropriate normal serum between
  certain steps may also help to reduce background.
• To avoid release of the blocking Fab antibodies
  by labelled secondary antibodies, the tissue or
  cells may be lightly fixed after the blocking
  step with a fixative, such a glutaraldehyde,
  provided that this fixation does not severely
  affect antigenicity of the second antigen to be
  labelled.

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Example A Use of conjugated Fab fragments for
labelling and blocking
1
2

• Incubate with the first, primary antibody.
• Incubate with an excess of Probe 1-conjugated Fab
  antibody against the host species of the primary
  antibody.
• Proceed with the labelling of the second antigen
  as usual.

3
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Example B. Use of unconjugated Fab fragments to
convert the first, primary antibody into a
different species.

• Incubate with the first, primary antibody.
• Incubate with an excess of unconjugated Fab
  fragment raised against the host species of the
  primary antibody.
• Incubate with Probe 1-conjugated tertiary
  antibody an anti-IgG (HL) or an anti-F(ab')2
  raised against the host species of the Fab
  fragment. The tertiary antibody must not
  recognise the host species of the either the
  primary antibodies or the second, secondary
  antibody.
• Incubate with the second, primary antibody.
• Incubate with Probe II-conjugated to the second,
  secondary antibody (that does not recognise the
  host species of either the Fab antibody or the
  tertiary antibody).

2
1
3
4
5
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Example C. Use of unconjugated Fab fragments for
blocking after the first, secondary antibody
step.
2
1

• Incubate with the first, primary antibody.
• Incubate with Probe I-conjugated to the secondary
  antibody.
• Incubate with normal serum (as a source of
  non-immune IgG) from the same host species as the
  primary antibodies, to saturate any open antigen
  binding sites on the first secondary antibody so
  it cannot bind the second, primary antibody.
• Incubate with an excess of unconjugated Fab
  antibody against the host species of the primary
  antibody. The Fab antibody should come from the
  same host species as the conjugated, secondary
  antibody.
• Incubate with the second, primary antibody.
• Incubate with Probe II-conjugated to the same
  secondary antibody as used in step 2.

4
3
6
5
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The Mouse on Mouse Question
1
2
X

• If one wishes to detect a mouse monoclonal on
  mouse tissue (that may have some mouse IgG
  present)
• Block with normal Goat serum
• Apply unconjugated Fab anti-mouse IgG (HL) to
  block any mouse IgG that may be present.
• Apply mouse primary antibody
• Apply conjugated goat anti mouse as the secondary
  antibody.

3
4
X
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Fluorophores

• The selection of fluorophores depends on
• Instrument set-up. For example, availability of
  light sources, filter sets, and detection
  systems.
• Degree of colour separation desired for multiple
  labelling. For example, Rhodamine Red-X and Texas
  Red give better separation from fluorescein than
  tetramethyl Rhodamine.
• Sensitivity required. For example, Cy3 and Cy5
  are brighter than other fluorophores.

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Table 1 Approximate peak wavelengths of
absorption and emission for different
fluorophore-conjugated, affinity-purified
antibodies.
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Excitation and emission spectra of different
fluorophore conjugated, affinity-purified
antibodies.
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Aminomethylcoumarin acetate (AMCA)

• Protein conjugates of AMCA absorb light maximally
  near 350 nm and fluoresce maximally near 450 nm.
• For fluorescent light microscopy, AMCA can be
  excited with a mercury lamp and observed using a
  UV filter set available from most microscope
  manufacturers.
• Since blue fluorescence is more difficult for the
  human eye to see than other colours,
  AMCA-conjugated, secondary antibodies should be
  used with the most abundant antigens in
  multiple-labelling experiments.

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Fluorescein Isothiocyanate (FITC)

• This dye absorbs light maximally at 492 nm and
  fluoresces at 520 nm. The only major disadvantage
  of FITC is its photobleaching (fading), which may
  be reduced in the presence of an anti-fading
  reagent such as n-propyl gallate.
• DTAF-conjugated Streptavidin is brighter than
  FITC-conjugated Streptavidin. Fluorescence from
  many fluorophore-conjugated streptavidins and
  egg-white avidins in solution are enhanced by the
  addition of a saturating amount of free biotin.
  Particularly the difference between DTAF- and
  FITC-conjugated streptavidins.
• Fluorescence from FITC-streptavidin is extremely
  low when no biotin is bound to the molecule.
  However, after addition of free biotin, there is
  a 16-fold increase in fluorescence. A similar
  response from DTAF-streptavidin was less dramatic
  (a 1.9-fold increase).

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Cyanine Dyes - Cy2, Cy3, and Cy5

• These cyanine dyes represented the beginning of a
  new generation of fluorophores, originally coming
  from the laboratory of Dr. Alan Waggoner at
  Carnegie-Mellon University.
• Cyanine dyes are
• much brighter,
• more photostable,
• and give less background than most other
  fluorophores.

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Cyanine Dyes - Cy2

• Cy2 fluoresces in the green (510 nm) like FITC
  (520 nm), (use existing FITC filter sets)
• Is more photostable
• Less sensitive to pH
• More fluorescent in organic mounting media
• Cy2 may, therefore, be visualised for longer
  times in the microscope
• May appear to be brighter than FITC without the
  use of anti-fading agents added to aqueous
  mounting media.

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Cyanine Dyes - Cy3

• Maximally excited near 550 nm with peak
  fluorescence near 570 nm.
• For fluorescent light microscopy, it may be used
  with traditional tetramethyl Rhodamine (TRITC)
  filter sets, as their excitation and emission
  spectra are nearly identical.
• Can be excited to about 50 of maximum with the
  514 nm or 528 nm lines of an Argon ion laser, or
  to about 75 of maximum with a Helium-Neon laser
  (543 nm line) or mercury lamp (546 nm line).
• Can be used with fluorescein for double
  labelling however, the use of narrow band-pass
  filters is recommended due to the overlap in
  fluorescence.
• Can also been paired with Cy5 for multiple
  labelling using a confocal microscope equipped
  with a Krypton/Argon laser and a far-red detector
  (e.g. a CCD).

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Cyanine Dyes - Cy5

• Excited maximally near 650 nm and fluoresces
  maximally near 670 nm. Can be excited with a
  Krypton/Argon laser (98 of maximum with the 647
  nm line) or a Helium/Neon laser (63 of maximum
  with the 633 nm line).
• Can been used with a variety of other
  fluorophores for multiple labelling due to a wide
  separation of its emission from that of
  shorter-wavelength-emitting fluorophores.
• Major advantage - lower autofluorescence of
  biological specimens from the red light used to
  excite other fluorophores.
• Due its emission maximum at 670 nm, Cy5 cannot be
  seen well by eye, and therefore cannot be used
  with conventional epifluorescence microscopes. It
  is most commonly visualised with a confocal
  microscope equipped with the proper laser for
  excitation (e.g., a Krypton/Argon) and a far-red
  detector (e.g., a CCD).

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Cyanine Dyes - Cy2, Cy3, and Cy5

• Anti-fading agents are not usually required when
  visualising cyanine dye conjugates in an
  epifluorescence microscope, but should be added
  to aqueous mounting media for confocal laser
  scanning microscopy.
• It is important to avoid the use of mounting
  media containing any aromatic amines, such as
  p-phenylenediamine which can react with cyanine
  dyes (especially Cy2) and cleave away half of the
  molecule, resulting in weak and diffused
  fluorescence after storage of stained slides.
  Other anti-fading agents, such as n-propyl
  gallate, may be used for mounting cyanine dye
  stained sections in aqueous media.
• Organic based mounting media, such as DPX or
  methyl salicylate, also may be used for cyanine
  dyes. DPX will harden into a plastic-like
  permanent medium, whereas methyl salicylate is a
  liquid so the cover slip needs to be sealed to
  prevent evaporation.

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Cyanine dyes
A fluorescent confocal photomicrograph of an
astrocyte from a rabbit optic nerve labelled with
monoclonal glial fibrillary acidic protein
antibody and detected with Cy3-conjugated
anti-mouse IgG (HL). Photo contributed by Scott
Rogers, Joseph Ghilardi, and Patrick Mantyh,
Dept. of Psychiatry, University of Minnesota.
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Cyanine dyes
A fluorescent confocal photomicrograph of a rat
spinal cord neuron labelled with rabbit
polyclonal substance preceptor antibody in
conjunction with Cy3-conjugated anti-rabbit IgG
(HL). Image contributed by Scott Rogers,
Joseph Ghilardi, and Patrick Mantyh, Dept. of
Psychiatry, University of Minnesota
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Artery in Human Skin

• Montage of six 20X images of triple-stained
  artery in human skin acquired on a CARV non-laser
  confocal microscope. Nerves are stained
  orange-red with a Cy3-secondary antibody used to
  detect anti-Protein Gene Product 9.5. Type IV
  collagen in basement membrane is localized with
  Cy2-secondary antibody (green) and Cy5-Ulex
  Europeaus Agglutinin type I (pseudo-colored blue)
  is used to stain endothelial cells.
• Photo contributed by Dr. William R. Kennedy and
  Dr. Gwen Wendelschafer-Crabb, Department of
  Neurology, University of Minnesota.

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Confocal image of human skin innervation

• A skin biopsy of finger, 100µm section,
  immunostained for pan-neuronal marker, protein
  gene product 9.5, localized with Cy3 (red and
  yellow) and basement membrane marker, type IV
  collagen, with Cy2 (green). Epidermal nerve
  fibers arise from the nerve bundles comprising
  the subepidermal neural plexus. A Meisner's
  corpuscle (M) is present in the papillary dermis.
  Basement membrane labeling delineates the
  boundary between epidermis (E) and dermis (D) as
  well as capillaries (C) and a sweat gland duct
  (SD).
• Photo contributed by Dr. William R. Kennedy,
  Department of Neurology, University of Minnesota.
  Submitted by Dr. Gwen Crabb.

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Tetramethyl Rhodamine Isothiocyanate (TRITC),
Rhodamine Red-X (RRX), and Texas Red (TR)

• All three Rhodamine derivatives have different
  absorption (550 nm, 570 nm, and 596 nm,
  respectively) and emission (570 nm, 590 nm, and
  620 nm, respectively) maxima.
• Although TRITC has been used most commonly with
  FITC for double labelling, better colour
  separation is achieved by using Rhodamine Red-X
  (an improved form of Lissamine rhodamine B) or
  Texas Red, although the use of Texas Red may lead
  to slightly higher background staining.
• For double labelling in flow cytometry,
  phycoerythrin (instead of rhodamine) conjugates
  are recommended for use with fluorescein since
  both fluorophores can be excited by a single
  wavelength (488 nm) of light.

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Rhodamine Red - X

• Rhodamine Red-X, a red-fluorescing dye
  manufactured and patented by Molecular Probes,
  Inc., is a succinimidyl ester of Lissamine
  rhodamine B-labelled aminohexanoic acid.
• Rhodamine Red-X conjugates are superior to those
  of Lissamine rhodamine B in a number of respects.

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Rhodamine Red - X

• The reactive succinimidyl ester provides a more
  consistent conjugation and is less harmful to the
  activity and stability of proteins than the more
  highly reactive sulfonyl chloride on Lissamine
  rhodamine B reactive dye.
• Rhodamine Red-X conjugates also have a spacer arm
  (from aminohexanoic acid) which extends the dye
  out from the surface of the protein.
  Consequently, proteins conjugated with Rhodamine
  Red-X are significantly brighter than those
  conjugated with Lissamine rhodamine B sulfonyl
  chloride.

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Rhodamine Red - X

• Rhodamine Red-X is recommended for triple
  labelling using a confocal microscope with Cy2
  and Cy5 because the emission lies about midway
  between that of Cy2 and Cy5 with little overlap.
  And, the commonly used Krypton/Argon ion laser
  emits at 488 nm, 568 nm, and 647 nm, which are
  optimal for Cy2, Rhodamine Red-X, and Cy5,
  respectively.
• Rhodamine Red-X may also be used for double
  labelling with Cy2 or for triple labelling with
  Cy2 and AMCA with an epi-fluorescent microscope
  using a mercury vapour lamp. (Cy 5 is not
  recommended for conventional epi-fluorescence
  microscopy because mercury vapour lamps do not
  emit any lines of light between 620 nm and 705
  nm. Furthermore, the fluorescence emission from
  Cy5 is not very visible to the human eye.)

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