Announcements

1
Announcements

• Reports posterized and missing images.
• Remember to close shutter on epifluorescence.
• Please log in and out of the confocal log book.
• Dont make me have to penalize you by losing
  points!
• Also log in the general use sheet, so the
  Microscopy facility can justify itself to the
  MAN.
• Finally, sign up for TBA time or other time when
  you want to use the scope to reserve your spot.
• Be thinking about your projects.
• I have the Carolina Biologicals catalog for
  source of material.
• TBA group 1 may need to come at different time
  this week.

2
Immunolabeling

• General problems
• Immunolabeling
• General considerations
• Trouble-shooting
• Controls for multiple antibody labeling
• Filters for fluorescence
• Demo and TBA

3
Problem 1 Bad DIC

• If you dont see a good DIC effect, first check
  that everything is set for DIC
• Both polarizers in
• Both Wollaston prisms in
• Kohler illumination set up
• The knob on the second prism adjusted to neutral
  gray
• If you still dont have good DIC, then try this
• Pull out the prisms so you have polarization
  setup
• Check that you have extinction (black background)
• If not, then adjust bottom polarizer so that it
  is 90o to the top polarizer.
• But the prisms back in and you should have nice
  DIC

4
Problem 2 Posterization of images

• Java tutorial http//micro.magnet.fsu.edu/primer/
  java/digitalimaging/processing/bitdepth/index.html
  
• When describing digital images, gray-level
  resolution is a term that refers to the number of
  shades of gray utilized in preparing the image
  for display. Digital images having higher
  gray-level resolution are composed with a larger
  number of gray shades and are displayed at a
  greater bit depth than those of lower gray-level
  resolution.
• An over-enthusiastic levels adjustment with
  Photoshop will also do this.
• Oshel Bringing down the "white" (far right)
  arrowhead (so perhaps also bringing up the
  "black" far left arrowhead) too much posterized
  the image. Looks like the bit-depth is getting
  truncated.
• Check your images in Photoshop resave with no
  levels adjustment if you see posterization.

5
Microtubules (Anti-tubulin)
Microtubules of bovine pulmonary artery
endothelial cells tagged with antibovine
alpha-tubulin mouse monoclonal 236-10501
(A-11126) and subsequently probed with Alexa
Fluor 488 goat antimouse IgG (HL) antibody.
6
Making antibodies Monoclonals versus polyclonals

• Polyclonal antibodies bind to many sites on the
  antigen
• Typically made in rabbit, rat or other
• Monclonal antibodies bind to only one site on
  antigen
• Always made in mice

7
Antibody structure
150 kD glycoprotein
8
Antibody classes
Antibody Human and Mouse Human and Mouse Human and Mouse Human and Mouse Human and Mouse Human and Mouse
  Light Chain Subtype Heavy Chain                                                                                                                                                                                                                                                                                                                                                               
IgA    or        or     IgA1IgA2    1    2                                                                                                                                                                                                                                                                                                                                                               
IgE    or     None                                                                                                                                                                                                                                                                                                                                                                   
IgD    or     None                                                                                                                                                                                                                                                                                                                                                                  
IgM    or     None µ                                                                                                                                                                                                                                                                                                                                                               
  Human Human Human Mouse Mouse Mouse
IgG Light Chain Subtype Heavy Chain Light Chain Subtype Heavy Chain
IgG    or        or        or        or     IgG1IgG2IgG3IgG4    1    2    3    4    or        or        or        or     IgG1IgG2aIgG2bIgG3    1    2a    2b    3
9
ImmunolabelingProcedure

• Specific antibodies used to visualize protein
  distribution.
• Direct specific antibody tagged with
  fluorochrome.
• Indirect primary (specific) antibody unlabeled,
  secondary antibody w/fluorochrome.
• Why?

10
Immunolabeling References

• Harlow, E. and Lane, D. (1999). Using antibodies
  a laboratory manual. New York Cold Spring
  Harbor Press.
• Harlow, E. and Lane, D. (1988). Antibodies a
  laboratory manual. New York Cold Spring Harbor
  Press.
• Hibbs, A.R. (2004). Confocal microscopy for
  biologists. Kluwer Academic.
• Jackson ImmunoResearch Laboratories, Inc.
  www.jacksonimmuno.com

11
Major constraints to immunolabeling

• Local antigen concentration
• Large number locally
• Identical antigen-binding sites
• Modification of the antigen by fixation
• Immobilization without change in antigen
• Antibody access to the antigen
• Permeability of tissue, masking of epitopes
  (antibody-binding site on antigen)
• Antibody specificity

12
Fixation for immunolabeling
Fixative Advantages Disadvantages
Methanol, 100, -20oC Excellent structure of cells, required for microtubule preservation Masks some antigenic sites, shrinkage, permeabilization with detergent necessary, not effective for phalloidin staining
Acetone, 100, -20oC Good antigen preservation, good permeabilization, low background fluorescence Poor structural integrity of cells, severe shrinkage and flattening
Formaldehyde, 2-4, RT or 4oC Commercial grade may contain MeOH, quick penetration Slow polymerization, permeabilization with detergent necessary
Paraformaldehyde, 2-4, RT or 4oC Excellent antigen preservation, low background Degrades quickly at RT
Gluteraldehyde, 3, RT or 4oC Best structural preservation Destroys most antigen sites, high background fluorescence
13
Permeabilizition

• Allows penetration of large antibody molecules
  into the cellular tissue.
• Typical non-ionic detergents used at 0.05-0.1 in
  buffers such as phosphate-buffered or
  Tris-buffered saline (PBS or TBS)
• Triton X-100
• Tween 20
• NP-40
• Exoskeletons or other extracellular structures
  may require other chemical or physical
  disruption.
• E.g. chitinous exoskeletons can be permeabilized
  by sonication.

14
Sectioned samples

• Paraffin-embedded, sectioned samples
• Usually not necessary for confocal
• Plant tissues sometimes prepared this way for
  confocal
• Limitation of about 200 µm for light penetration.
• Cryo-sectioned samples
• Sometimes the only way to preserve antigenic
  sites.

15
Methods of immunolabeling

• Whole mount
• Processing is done in small tubes or multi-well
  plates.
• Adhesion of sample to slides, using poly-L-lysine
  or by fixation.
• Spread of solutions can be limited by drawing
  rings with PAP pen or by using special slides.
• Humidity chamber necessary to prevent drying out.

16
Blocking agents to prevent non-specific antibody
binding

• Bovine serum albumin (BSA), 0.5-2
• Skim milk, 5
• Serum (1-10) from the same species used to raise
  the secondary antibody (usually goat or donkey).
• Dissolved in buffer, sample treated before
  addition of primary antibody.
• Antibody solutions usually contain blocking agent
  as well.

17
Testing specificity of a new antibody

• Try antibody in immunoblotting (denatured
  epitope) or immunoprecipitation (native epitope)
  experiments to look for specific and
  side-reactions.
• Perform appropriate controls
• Negative control confirms that a positive result
  in not artifactual
• Preimmune or normal serum substituted for primary
  antibody
• Secondary antibody on its own
• Positive control confirms that a negative result
  is not due to poor technique or reagents
• Test against original target if attempting
  cross-reactivity
• Confirm staining pattern with antibody to another
  epitope of the antigen.
• If available, compare staining pattern in
  wildtype versus deletion mutation.

18
Variations of indirect immunolabeling
Streptavidin- fluorochrome
Secondary- fluorochrome
Streptavidin- fluorochrome
Secondary -biotin
Primary- biotin
Primary
Primary
An enzyme, e.g. horseradish peroxidase or
alkaline phosphatase, can also be substituted for
the fluorochrome. In this case, detection is by
conversion of a substrate to a colored product.
19
Biotin-streptavidin
BREAK Start Staining
20
Immunolabeling of Drosophila embryos (Rothwell,
and Sullivan, 1998. In Drosophila Protocols,
Sullivan, W. Ashburner, M. and Hawley, R.S.
(eds.) Cold Spring Harbor Press, pp. 141-157)
Engrailed antibody, Drosophila embryo
21
PBTA (1X PBS, 1 BSA, 0.05 Triton X-100, 0.02
Sodium Azide)

• 10X PBS is
• NaCl 80 g
• KCl 2 g
• Na2HPO4 14.4 g
• KH2PO4 2.4 g
• Dissolve all components in 800 ml H2O. Adjust
  the pH to 7.4 with HCl. Sore at RT.
• PBTA solution
• Mix the following components
• 10X PBS 50 ml
• BSA 5 g
• Triton X-100 250 ul
• Sodium azide 0.1g
• Adjust volume to 500 ml with H2O.

22
Immunolabeling Day 1

• Embryos have been fixed with formaldehyde and
  stored in methanol at -20oC.
• Remove as much of the methanol as possible.
• Add 500 µl PBTA solution. Allow embryos to
  rehydrate in this solution at room temperature
  for 15 minutes on a rotator.
• Remove the PBTA and add 250 µl diluted primary
  antibody (in PBTA). Incubate on a rotator
  overnight at 4oC.
• 15 engrailed, 15 even-skipped, 125 tubulin
• Controls (a) 2o antibody only, (b) neither
  antibody.

23
Immunolabeling Day 2(In Microscopy facility)

• Remove the primary antibody and rinse the embryos
  3X with PBTA, allowing the embryos to settle
  between rinses. Wash the embryos for at least 1
  hr at RT on a rotator. Longer washes and more
  rinses usually produce cleaner images.
• Add fluorescently labeled secondary antibody (in
  fridg), diluted 1250 in PBTA (250 µl total
  volume) and incubate 1 hr at RT on a rotator.
• Remove the secondary antibody. Wash 3X with PBTA
  as in step 1 above.
• You can cheat and wash for a total of 30 minutes.
• Rinse the embryos 4X in PBS-Azide to remove the
  detergent.
• You can cheat and do 2X washes

24
Mounting and Storage of Embryos

• Remove as much of the PBS-Azide as possible and
  add 40 µl glycerol-based mounting medium (90
  glycerol 10 PBS containing 10 mg/ml N-propyl
  gallate to reduce photobleaching in freezer).
• Gently resuspend and transfer embryos in mounting
  medium to a slide using a P-200 pipetman with
  yellow tip cut at an angle to allow pipeting of
  viscous solution.
• 40 µl is ideal for 22 X 22 coverslip
• Place a coverslip over the embryos and seal with
  nail polish (sealing is optional).
• Store slides flat at -20oC in the dark.

25
Reports (due Feb. 20)

• Include
• The technical information on fluorescent probe
  and image collection, as before.
• Methods reference to Rothwell and Sullivan
  (1998).
• Interpretation of the image, including embryonic
  stage, cellular and sub-cellular localization
  (e.g. in nuclei of dorsal epithelial cells at
  extended germ band stage).
• On reserve Lawrence (1992), Gilbert (2000) for
  staging embryos.