Announcements• Reports: posterized and missing images.• Remember to close shutter on epifluorescence.• Please log in and out of the confocal log book.

– Don’t 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.

Immunolabeling

I. General problems

II. ImmunolabelingA. General considerations

B. Trouble-shooting

C. Controls for multiple antibody labeling

III. Filters for fluorescence

IV. Demo and TBA

Problem 1: Bad DIC

• If you don’t 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 don’t 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

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.

Microtubules (Anti-tubulin)

Microtubules of bovine pulmonary artery endothelial cells tagged with anti–bovine alpha-tubulin mouse monoclonal 236-10501 (A-11126) and subsequently probed with Alexa Fluor 488 goat anti–mouse IgG (H+L) antibody.

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

Antibody structure

150 kD glycoprotein

Antibody classesAntibody Human and Mouse

  Light Chain Subtype Heavy Chain

                                                                                           

                         

IgA    or        or    

IgA1

IgA2    1

   2

IgE    or     None    

IgD    or     None   

IgM    or     None µ

  Human Mouse

IgG Light Chain Subtype Heavy Chain Light Chain Subtype Heavy Chain

   or        or        or        or    

IgG1

IgG2

IgG3

IgG4

   1

   2

   3

   4

   or        or        or        or    

IgG1

IgG2a

IgG2b

IgG3

   1

   2a

   2b

   3

ImmunolabelingProcedure

• Specific antibodies used to visualize protein distribution.

• Direct: specific antibody tagged with fluorochrome.

• Indirect: primary (specific) antibody unlabeled, secondary antibody w/fluorochrome.– Why?

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

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

Fixation for immunolabelingFixative 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

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.

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.

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.

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.

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.

Variations of indirect immunolabeling

Streptavidin-fluorochrome

Primary

Secondary-fluorochrome

Primary

Secondary-biotin

Primary-biotin

Streptavidin-fluorochrome

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.

Biotin-streptavidin

BREAK: Start Staining

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

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.

Immunolabeling: Day 1

1. Embryos have been fixed with formaldehyde and stored in methanol at -20oC.

2. Remove as much of the methanol as possible.3. Add 500 μl PBTA solution. Allow embryos to

rehydrate in this solution at room temperature for 15 minutes on a rotator.

4. Remove the PBTA and add 250 μl diluted primary antibody (in PBTA). Incubate on a rotator overnight at 4oC.

• 1:5 engrailed, 1:5 even-skipped, 1:25 tubulin• Controls: (a) 2o antibody only, (b) neither antibody.

Immunolabeling: Day 2(In Microscopy facility)

1. 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.

2. Add fluorescently labeled secondary antibody (in fridg), diluted 1:250 in PBTA (250 μl total volume) and incubate 1 hr at RT on a rotator.

3. Remove the secondary antibody. Wash 3X with PBTA as in step 1 above.

• You can cheat and wash for a total of 30 minutes.

4. Rinse the embryos 4X in PBS-Azide to remove the detergent.

• You can cheat and do 2X washes

Mounting and Storage of Embryos

1. 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).

2. 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

3. Place a coverslip over the embryos and seal with nail polish (sealing is optional).

4. Store slides flat at -20oC in the dark.

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.