Antibody Staining with Tyramide Enhancement

The Renaissance TSA-Indirect kit (catalogue # NEL700A from New England Nuclear) specifically amplifies a peroxidase signal, permitting the use of a lower concentration of primary antibody. This gives an improved signal:noise ratio for antibodies that aren't great, and allows you to use a higher dilution for antibodies that are precious and irreplaceable. You will need to try several concentrations of a familiar antibody to find the one that works well with tyramide enhancement; try 1:5 and 1:25 dilutions from what you usually use.

Fixation:

  1. Transfer embryos from a molasses plate to a basket and rinse extensively with distilled water to remove yeast paste.
  2. Dechorionate by immersing in 50% Clorox bleach for 5 min at room temp.
  3. Rinse with 0.1% Triton X-100 to remove Clorox, then rinse with distilled H2O to wash away detergent. Blot on paper towel.
  4. Use a fine paintbrush to transfer embryos to 5ml 1:1 heptane: formaldehyde sol'n (see recipe) in a glass scintillation vial. A single layer of embryos is desired at the interface. (Perform this step quickly because dechorionated embryos will dry out).
  5. Secure vial on its side on a platform shaker and shake at moderate speed (125 rpm) for 15-20 min at room temp (shorter times are better for antigens that are particularly sensitive to fixatives).
  6. Draw off embryos from the interface using a Pasteur pipet. Discard the lower aqueous phase and transfer embryos in heptane to a 1.5 ml Eppendorf tube.
  7. Rinse embryos 3 times with 1 ml heptane. This is best accomplished by sucking the embryos into a Pasteur pipet with the heptane, then discarding any residual formaldehyde as in step 6. The embryos can be left in the same Eppendorf tube and the heptane removed after each wash. Complete steps 6 and 7 quickly--embryos should not sit in formaldehyde longer than necessary.
  8. Add 0.6ml of heptane followed by 0.6 ml of methanol to each tube. Hold tubes in fist and shake vigorously for 30-60 sec to facilitate devitellinization of the embryos. Devitellinized embryos sink to the bottom of the tube. Remove the top heptane layer and the interface, which will contain empty vitelline membranes and embryos which are not devitellinized.
  9. Rinse embryos several times with 1 ml methanol to remove remaining vitelline membranes.
  10. Store embryos at -20°C for future staining.

Staining:

  1. Transfer an appropriate number of embryos for staining into new Eppendorf tubes (this will be approximately 25-50ml settled volume of embryos). Draw off methanol from surface of embryos.
  2. Add 1 ml fresh methanol to each tube. Add 50 microliters 30% H2O2 to each tube and rock at room temperature for 20 minutes.
  3. Remove peroxide mixture and add 1 ml of 100% methanol to each tube. Rock at room temp for 5 minutes. Repeat.
  4. Add 1 ml of 50% methanol to each tube and rock at room temp for 5 min.
  5. Remove methanol and rinse 4-5 times with 1 ml PBT. Rock at room temp for 5 min between washes.
  6. Remove PBT and add 1ml PBT + 2% goat serum (Vector Laboratories) to each tube. Block at room temperature for at least 1 hour with rocking.
  7. Dilute primary antibody to desired concentration. Transfer embryos into 0.5 ml Eppendorf tubes. Remove excess block, without creating bubbles. Add 75-150 microliters of primary antibody to each tube, rock at room temp for 1-2 hours, then rock at 4°C overnight.
  8. Remove all primary antibody and save at 4°C (depending on antibody titre and abundance of antigen, can re-use multiple times). Transfer embryos to 1.5 ml Eppendorf tubes. Wash 8 times with 1 ml changes of PBT for 5-8 min each (rocking at room temp).
  9. Block with PBT + 2% goat serum as in step 6. Rock at least 1 hour at room temp.
  10. Prepare pre-adsorbed secondary antibody according to recipe. Remove block and add 75-150 microliters of secondary antibody. Rock at room temp for 45 min. Remove all secondary antibody and save for re-use.
  11. Wash embryos 8 times with PBT as in step 8.
  12. Block with PBT + 2% goat serum as in step 6. Rock for 30 min at room temp. (This step can go overnight at 4°C.)
  13. Make A+B solution in block (PBT + 2% goat serum--see recipe). This solution should sit at room temp for 30 min before use. Remove block from embryos, add 500 microliters of A+B solution to each tube, and rock at room temp for 1/2 hour.
  14. Wash 8-10 times with PBT as in step 8.
  15. Prepare a working solution of BT (biotinyl tyramide--see recipe). Remove block and add 150 microliters of BT. Rock for 20 min at room temp.
  16. Wash 8-10 times with PBT as in step 8.
  17. During the 5th or 6th wash, prepare A+B solution in PBS (see recipe). Once again, this needs to sit at room temp for 30 min before use. Remove PBT from embryos and add 0.5 ml of A+B solution to each tube and rock at room temp for 1/2 hour.
  18. Wash 5X with PBT, rocking at room temp for 5 min between each wash.
  19. Prepare DAB solution (see recipe). For blue stain, mix two parts DAB solution + one part 3% NiCl2. For orange-brown stain, use the DAB solution with no NiCl2.
  20. Remove last PBT wash from tube and add 0.5 ml of the appropriate DAB solution to each tube. Rock for at least 10 min at room temp.
  21. Transfer 100 microliters embryos in PBS/DAB to a glass dish with a blue pipet tip (wet tip with PBT first so embryos don't stick). Add ~5 microliters 0.3% H2O2 (freshly diluted from 30% stock into dH2O). It may take seconds to several minutes for staining to occur, depending on the abundance of the antigen and the quality of the antibody. Observe the extent of staining under a stereomicroscope and, when the color is appropriately developed, stop the reaction by washing with several changes of PBT. (Note: dispose of all tips and Eppendorfs exposed to DAB in special beaker. DAB must be deactivated with bleach because it is extremely mutagenic.)
  22. Stained embryos can be cleared prior to mounting as follows: dehydrate through an ethanol series (30%, 50% and 70% sequentially) followed by 5-6 changes of 1 ml 100% ethanol, then 2 changes of 1 ml methyl salicylate (rock at room temp for 5 min between washes).
  23. Mount embryos in DPX (Fluka). Photograph using Kodak EPT 135-36 film at ASA 160 or Kodak Tech Pan (TP 135-36) at ASA 50.

Embryos can be double stained with two different antibodies. After stopping the first staining reaction with several changes of PBT, block again with 2% goat serum in PBT for one hour. Add the second primary antibody, let rock at room temp for one hour, then transfer to 4°C to rock overnight. Repeat the washing and secondary antibody steps, then stain again. By omitting the NiCl2 in one of the staining reactions, you can stain with different colors for the different antibodies (see above). Staining blue for the first antibody and orange-brown for the second works well.

Recipes:

Fix solution:

1.375 ml dH2O
0.5 ml 5X PEM
0.625 ml 16% formaldehyde (EM grade from Polysciences)
2.5 ml heptane

Combine first three ingredients, then add the heptane and shake to saturate the heptane with formaldehyde.

5X PEM:

0.5 M Pipes, pH 6.9
10 mM MgSO4
5 mM EGTA, pH 8.0

0.2% PBT:

500 ml 1X PBS
1.0 ml Tween-20

Blocking Solution, per ml:

980 microliters PBT
20 microliters goat serum (Vector Laboratories)

Secondary antibody:

440 microliters PBT
10 microliters goat serum
50 microliters 1:50 pre-adsorbed biotinylated secondary antibody (e.g. if the primary antibody is a rabbit serum, use goat anti-rabbit IgG. Various biotinylated secondary antibodies are available from Vector Labs. See below for pre-adsorption.)

A+B solution:

VectaStain Elite ABC kit from Vector Labs, one reaction volume
480 microliters Blocking Solution (for first A+B incubation) or PBS (for second A+B incubation)
10 microliters reagent A
Vortex well, then add:
10 microliters reagent B
Vortex well, let stand at room temp for 30 minutes before using.

BT Working Solution:

For one reaction volume:
147 microliters Amplification Diluent (provided)
3 microliters Biotinyl Tyramide Reagent
BT reagent is in DMSO, so thaw thoroughly before using and be careful.

DAB Solution:

0.5 mg/ml diaminobenzidine in PBS (not PBT)

A 2X stock of DAB in PBS can be aliquoted and stored at -20°C but, after thawing, it should be vortexed and then centrifuged thoroughly to remove the insoluble oxidation product that will otherwise cause bad background.

Pre-adsorption of antibodies:

  1. Fix an overnight collection of wild type embryos, as described above. (If pre-adsorbing an antibody directed against an antigen expressed in embryos, must use embryos representing stages during which this expression does not occur).
  2. Re-hydrate embryos from 100% methanol by washing 5 times with 1 ml PBT. Use 100-150 microliters settled volume of embryos.
  3. Add to embryos:
    1440 microliters PBT
    30 microliters goat serum (2% final concentration)
    30 microliters antibody stock (=1:50 dilution)
  4. Rock at 4° overnight.
  5. Store the pre-adsorbed antibody at 4°C. For long-term storage, it may be desirable to add 0.02% sodium azide to inhibit bacterial growth.