LM Immunolabeling follows a protocol similar to well established indirect methods. With immunogold labeling, however, there are no hazardous materials required. Furthermore, the method is a simple 2-step incubation which gives a more intense stain than conventional peroxidase or PAP techniques. This permits much higher dilutions of primary antibody and gold conjugate, thus it is more economical and results in lower background. The following protocol is useful for staining embedded or frozen sections, suitably treated cell smears mounted on glass slides, or cells in culture. Many variations have been previously published, but the basic principle applies to all.

Immunolabeling Procedure for Light Microscopy

  1. Dewax paraffin sections and wash in PBS (buffer 1) (10 min).
  2. Place a droplet (e.g. 50 µl) of heat-inactivated neat normal goat serum on the dewaxed or frozen section and incubate for 15 min (not for Protein A).
  3. Shake off the excess goat serum and place a 50 µl (or less) droplet of diluted primary antibody on the section. Incubate for 30-60 min at room temperature, or overnight at 4oC.
  4. Wash slides thoroughly in several changes of PBS (e.g. pipette rinsing followed by 5 x 1 min immersion in a 40-75 ml Coplin jar) to remove unbound antibody.
  5. Place 50 µl of diluted gold conjugate on the section and incubate for 1 hour.
  6. Wash thoroughly in several changes of deionized or distilled water (e.g. 5 x 1 min) to remove all unbound gold particles. All glassware must be ultraclean and water must be pure for this and each of the following steps.
  7. Immerse the slides in fresh silver enhancement solution (ask about our SilvEnhanceTM Kit) for 5-15 min. Wash thoroughly under tap water and counterstain lightly (e.g. hematoxylin or Nuclear Fast Red).

Immunolabeling Controls

A number of negative controls should be performed to confirm the specific labeling of the sections. Here are a few suggestions. Where possible, a known positive control should be included in the series to demonstrate the validity of the primary antibody and of the method.

  • Omit the first antibody and replace with buffer only.
    OR:
  • Replace the primary antibody with non-immune serum (rabbit or mouse), preferably from the same animal before immunization.
    OR:
  • Replace the primary antibody with a control antibody from the same species raised against an antigen known to be absent in the tissue.
    OR:
  • Absorb the primary antibody onto its antigen by addition of approximately 1 nM of antigen for 1 hour before the incubation.

Troubleshooting

During the silver enhancement step, development may be monitored by brief observation under normal lighting. Experience will determine the time required for this step. Underdevelopment produces a light brown stain, rather than intense black. Overdevelopment produces increased background, and a spreading of the black silver stain. The correct use of positive and negative controls is the same as the EM immuno-labeling procedures. Many causes and remedies for poor labeling are given here.

Result: No label

  • The antigen may be destroyed by preparative procedures. Use a different procedure (e.g. fixatives, cryosections, resins). Check that the temperature of tissue during embedding does not rise above 60°C (If above 60oC, max. 1 hr).
  • The antigen may be present in genuinely low amounts. Use longer incubation times and more concentrated primary antibody.
  • The primary antibody may be bad due to poor titer, age, improper storage, improper dilution, excessive freezing and thawing, etc. Run a positive control to check.
  • The pH of solutions may be excessively acidic or alkaline. Check the pH.
  • The silver enhancement procedure may be incomplete (stain is light brown or absent). Use longer incubation time with enhancement solution. Check positive controls.
  • Heavy counterstaining may mask silver stain. Use less counterstain.
  • Pretreat sections with silvenhance buffer 1-3 min, then apply equal amounts of Silver solution.

Result: Excessive Background Staining

  • Ionic concentration of solutions may be too low. Use increased salt concentration (up to 2.5%). Add ovalbumin, BSA or normal goat serum (not for Protein A) to approximately 1% in incubation solutions.
  • Sections may have been inadequately washed between incubations. Wash thoroughly.
  • Non-specific charge attraction of antibody can cause background Use 1% detergent (e.g. TWEEN 20) in all solutions. Include normal goat serum (not for Protein A) in all solutions. Increase concentration of normal goat serum before primary antibody incubation.
  • Free aldehyde groups in fixed tissue may be a source of background. Reduce by exposing sections to 0.5 M ammonium chloride for 1 hour before incubations.
  • The primary antibody concentration may be too high. Dilute by orders of magnitude.
  • The gold conjugate concentration may be too high. Dilute further.
  • Exposure to silver enhancement solution may be excessive (especially where antigen concentration is low). Reduce development time.
  • Silver may precipitate due to ionic impurities in the water. Wash thoroughly with distilled, deionized water. Make certain that glassware is very clean.
  • Silver may nucleate spontaneously on endogenous metals in tissue (Zn, Fe, Cd, etc.) Check negative controls.
  • Use fresh tissue whenever possible. Autopsy/old tissue tends to give background