Announcements, Week 6

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Announcements, Week 6

• Happy Darwin Day! (b. Feb. 12, 1809)
• BPAE cell reports due today.
• Paper discussion (Becky) Garcia-Pichel et al.
  2001
• TBA this week Drosophila embryos, mouse
  intestine cryostat sections
• Reports due Feb. 19.
• TBA for Group 4 following lecture.
• Week 7, Feb. 19 Fluorescent probes, live cell
  labeling
• TBA onion epithelium, report due Feb. 26
• Paper discussion next week (Rachel) Tan et al.
  2005.
• Week 8, Feb. 26 Midterm exam on lecture, lab
  material microscope checkout (change from end of
  semester) during TBA time allow 30 minutes each
• Project descriptions also due.

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Lecture Outline

• Immunolabeling (contd)
• General considerations
• Autofluorescence and background
• Controls
• Fluorescence
• Defined
• Absorption and emission spectra
• Filters
• Cross-over (bleed-through) compensation
• TBA Drosophila and mouse intestine samples

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Detection Methods
Method Advantages Disadvantages Recommended for
Fluorescence High resolution Doubling labeling possible Staining live cells possible Requires special microscope High resolution studies Double labeling
Enzyme High sensitivity Only need bright field light microscopy Permanent Low resolution Endogenous enzyme activities Double staining difficult Substrate toxicity Low resolution studies Rapid antigen screens
From Harlow and Lane, 1999
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Antibody Choice
Polyclonal antibodies Monoclonal antibodies Pooled monoclonal antibodies
Signal strength Excellent Fair Excellent
Specificity Good, but some background Excellent, but some cross-reactions Excellent, by avoiding any antibodies with cross reactions
Good features Signal strength Specificity Signal strength and specificity
Bad features Background Often need to titre Lower signal strength Availability
From Harlow and Lane, 1999
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Immunolabeling of live cells

• Commonly used for surface antigens.
• Cellular endocytosis can be used to take up
  antibody.
• Streptolysin-O (SLO) can be used to permeabilize
  cells and retain viability.

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Troubleshooting

• Problem 1 No specific staining with single
  labeling
• Possible causes
• Antigen not cross reactive, destroyed or
  inaccessible
• Perform positive control, obtain fresh material,
  permeabilize
• Primary antibody dead or not concentrated enough
• Obtain fresh or increase concentration
• Secondary antibody dead or not concentrated
  enough
• Obtain fresh or increase concentration
• Protocol errors
• Review protocol, modify
• Problem 2 Artifactual staining do neg. controls.

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Autofluorescence

• Autofluorescence is caused by the intrinsic
  properties of some structures, independent of
  antibody labeling.
• Aromatic amino acids and other molecules
  containing ring structures
• Chitin, chlorophyll, collagen, elastin
• Often worse with shorter wavelength excitation
  that with longer.
• Aldehyde cross-linking (especially
  gluteraldehyde), methanol fixation
• Low level of autofluorescence may be helpful to
  see limit of cells or tissues.
• Negative control with no fluorescent probe
  determines location and limits of
  autofluorescence.

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Background staining

• Caused by binding of antibody.
• Nonspecific background Binding of antibodies by
  parts other than antigen-binding site
• Spin down prior to use to remove large particles
• Titrate concentrations to minimum
• Use blocking reagents, e.g. BSA, nonfat dry milk,
  normal serum from same species as labeled
  antibody
• Use detergent in all solutions
• Reduce incubation times, increase wash times and
  numbers
• Specific background Caused by antigen binding in
  side reactions
• Dilute primary antibody in 1 normal serum from
  same species as labeled antibody

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Controls for immunofluorescence labeling

• Spectral properties of the available dyes limit
  the experimental freedom.
• Often it is even difficult to clearly separate
  two fluorescence markers.
• With more markers, the problem grows increasingly
  complex.

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Crossover
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Controls for Double Labeling (Indirect
Immunofluorescence)

• Experimental
• Mouse Primary 1 anti-mouse IgG secondary-FL 1,
  e.g. mouse anti-tubulin goat anti-mouse-rhodamin
  e.
• Rabbit Primary 2 anti-rabbit IgG secondary-FL
  2, e.g. rabbit anti-actin goat
  anti-rabbit-fluorescein.
• Negative Controls for Primary Specificity
• Pre-Immune Serum or non-immune serum from mouse
  Anti-mouse IgG secondary antibody-rhodamine.
• Pre-immune Serum or non-immune serum from rabbit
  Anti-rabbit IgG secondary antibody-fluorescein.

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Controls for Double Labeling, Indirect
Immunofluorescence (contd)

• Negative Controls for Secondary Specificity
• Primary 1 Secondary 2, Primary 2 Secondary 1
• Look for secondaries that say no
  cross-reactivity
• Staining with one antibody and another
  non-antibody probe (e.g. rhodamine-phalloidin or
  Sytox Green) is a simpler matter.
• If you want to use two monoclonal antibodies, one
  solution is to conjugate fluorochromes to them
  directly, e.g.
• mouse anti-tubulin-rhodamine
• mouse anti-actin-fluorescein
• Sequential staining is also possible, fixing
  between steps.
• Or another way is

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Fab Fragments for Blocking and Double Labeling of
Primary Antibodies from the Same Host
Species(Jackson ImmunoResearch Laboratories,
Inc.)


Key Key
       Rabbit anti-Antigen X       
       Rabbit anti-Antigen Y     Fluorescein (FITC)
     Fab fragment Goat anti-Rabbit IgG (HL)     Rhodamine Red-X (RRX)
       Goat anti-Rabbit IgG (HL)
      

                                                                             
See alternative methods at www.jacksonimmuno.com
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Imaging Double-Labeled Samples

• Select dyes that that are well-separated in their
  absorption and emission spectra.
• E.g. use Texas Red (596-620) instead of rhodamine
  (550-580) with fluorescein (490-520).
• Be careful about turning up the gain you can
  make almost anything fluorescent.
• Compensate for fluorescence cross-over (discussed
  below).

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What is fluorescence?Single-photon excitation

• Where a molecule emits light at a specific
  wavelength when irradiated by light of a shorter
  wavelength.
• Jablonski diagram depicts molecular events of
  single-photon fluorescence

2. excited lifetime
3. emission
1. absorption
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Fluorescence Multi-photon excitation

• 2 longer (infrared) wavelength photons absorbed
  simultaneously, emitting a shorter wavelength
  photon.
• Advantage of multi-photon confocal microscope is
  that thicker samples can be penetrated, compared
  to UV or visible light.

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Absorption and Emission SpectraSpectral overlap
and Stokes shift

• Spectral overlap must be eliminated by filters,
  otherwise brighter excitation light will overwelm
  dimmer emission light.
• The bigger the Stokes shift, the easier it is to
  separate excitation from emission.

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Reduced quantum yield using sub-optimal
excitation wavelength

• Quantum yield is a measure of the efficiency of
  conversion of absorbed light into emitted
  fluorescence.
• Same green light is emitted from purple versus
  blue excitation, just dimmer with purple
  excitation.
• Local environment, e.g. protein conjugation, pH,
  can also affect absorption spectrum and therefore
  quantum yield.

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Filters and Dichroic Mirrors

• Dichroic mirrors
• Reflect some wavelengths
• Transmit other wavelengths
• Excitation and Emission (Barrier) filters
• Absorbs some wavelengths, transmits others
• short pass allows transmission below cutoff
• long pass allows transmission above cutoff
• narrow band pass allows a range to be transmitted

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Excitation filters, dichroic mirrors and emission
filters
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Absorption and emission spectra, FITC and
rhodamine, with long pass 510, 565 filters
550 580
490 520
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BA510IF, BA530RIF CH 1
FITC only
Rhodamine only or CH 2
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2-channel imaging, using long pass 510 short
pass 530 ( narrow band pass), long pass 565
filters
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Typical Crossover Problem FITC and rhodamine
(TRITC) emission spectra
570 cutoff Ch. 1 Ch. 2
Solution 1 Cut off FITC emission tail using
OFFSET in Acquire panel or attenuate laser power
Solution 2 Collect 2 channels sequentially
Bleed-through of FITC into Ch. 2
Bleed-through of TRITC into Ch. 1
400 500 600 700
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Fluorescence Tutorials from Invitrogen/Molecular
Probes

• Basic Fluorescence http//probes.invitrogen.com/r
  esources/education/tutorials/1Introduction/player.
  html
• Spectra http//probes.invitrogen.com/resources/ed
  ucation/tutorials/2Spectra/player.html
• Filters http//probes.invitrogen.com/resources/ed
  ucation/tutorials/3Light_Sources_Filters/player.ht
  ml

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(a-c) AlexaFluor 488 and Cy3 simultaneous
scanning live samples require (d-f) AlexaFluor
488 and Cy3 sequential scanning possible w/
fixed samples
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Sequential Scanning
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Java Tutorial Crossover compensation

• http//www.olympusconfocal.com/theory/bleedthrough
  .html
• Java tutorial http//www.olympusconfocal.com/java
  /crossoversimulator/index.html

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Minimizing crossover specimen labeling
precautions (Molecular Expressions)

• Choose fluorochromes with as widely separated
  spectra as possible.
• Adjust concentrations of fluorescent stains so
  that intensities are close to equal
• When selecting fluorescent probes for
  multiply-labeled specimens, the brightest and
  most photostable fluorophores should be reserved
  for the least abundant cellular targets.

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Minimizing crossover instrumental approaches
(Molecular Expressions)

• Absorption spectra are generally skewed towards
  shorter wavelengths whereas emission spectra are
  skewed towards longer wavelengths.
• For this reason, multicolor fluorescence imaging
  should be conducted with the reddest (longest
  wavelength peak emission) dye imaged first, using
  excitation wavelengths that are only minimally
  absorbed by the skewed spectral tails of the
  bluer dyes.

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Balancing emission intensities reduces much
crossover
Emission only
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Controls for Double Labeling

• Background control specimen without secondary
  antibody or fluorochrome
• Controls for autofluorescence
• Bleed-through controls specimens labeled with
  each fluorochrome separately. To determine
  maximum gain before bleed-through
• Image green-labeled sample w/488 in Ch. 1, look
  for cross-over in Ch. 2.
• Image red-labeled sample w/543 in Ch. 2, look for
  crossover in Ch. 1.
• Using these settings, image double-labeled sample
  (same stain concentrations as above) using
  sequential scan.

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Quantum Dots

• Quantum dots are semi-conductor nanocrystals
  coated with inert polymer to which biomolecules
  can be attached, e.g. antibody.
• Advantages
• Less photobleaching
• High quantum yield
• Narrow, symmetrical emission spectra means less
  spectral overlap.
• Various colors can be excited by same laser line

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TBA this week

• Use your stained fly slides to collect Z-series
  of (a) lower mag overview (lastname 4A), and (b)
  high mag detail (lastname 4B), (c) negative
  control (low mag, lastname 4C).
• Use references on reserve in library to identify
  stages and structures stained.
• Save images in Week 6 folder and turn in a report
  next Monday (see new format).
• Mouse intestine cryostat section (16 um)
• Collect 2-channel image using (a) simultaneous
  imaging (lastname 5A) and (b) sequential imaging
  (lastname 5B).
• Compare dual-labeled samples to controls
• Submit two reports, one for each sample.