Immunogold Labeling with BioSite Colloidal Gold Reagents

General Information
Electron Microscope Applications (EM grade)
Cationic Colloidal Gold
Light Microscopy Applications (LM Grade)

General Information

Buffer Preparation

The following are recipes for diluting buffers. These are very general and may be supplemented with additional BSA, NaCl, ovalbumin or detergent (Tween 20) as desired. Gold labeled antibodies are usually applied in the pH range 7.4-8.2.

A. Tris-Buffered Saline (TBS)

To 75ml distilled water, add the following:
0.242g (20mM) Tris (tris-hydroxymethyl-aminomethane)
0.13g (20mM) NaN3 (preservative)
0.9g (150mM) NaCl
0.1g Bovine serum albumin (BSA)
Bring volume up to 100ml with distilled water and make sure all components are well dissolved. Adjust pH to 8.2 (or as desired) with 0.1 N HCl or 0.1 N NaOH and microfilter.

B. Phosphate-Buffered Saline (PBS)

To 75ml distilled water, add the following:
0.148g (approx. 10.4mM) Na2HPO4 (anhydrous)
0.043g (3.2mM) KH2PO4 (anhydrous)
0.13g (20mM) NaN3 (preservative)
0.9g (150mM) NaCl
0.1g Bovine serum albumin (BSA)
Bring volume up to 100ml with distilled water and make sure all components are well dissolved. Adjust to pH 7.4 (or as desired) with 0.1 N HCl or 0.1 N NaOH and microfilter.
Dilutions

Primary antibody concentration should be determined by the investigator. Initial tests should include serial dilutions of the primary antibody to attain optimal labeling while keeping background at a minimum.

A. Microscopy

It has been found that dilutions between 1:10 and 1:100 (or greater) of the conjugate serve most applications. In general, high dilution of the gold conjugates, combined with longer incubation times, reduces the degree of non-specific attachment to highly charged components of the tissue matrix.

Extensive washing with buffer is recommended between incubation steps to further reduce non-specific attachment to the section. BioSite gold conjugates are supplied in Tris buffer, pH 8.2, containing 20% glycerol. Dilutions may be made in Tris, PBS, or other buffers at varying pH values.

B. Cationic Gold

The cationic conjugate is best applied directly to the section at a dilution of 1:10 to 1:100 (or greater) in a buffer such as Tris or PBS adjusted to the appropriate pH. The pH will determine the degree of labeling of the anionic sites. Low pH (e.g. pH 2 to pH 3) is likely to produce the most specific labeling with the lowest background, but this should be experimentally determined. The most specific labeling is observed at higher dilutions (around 1:100). An incubation of 30-60 minutes in a moist chamber will be sufficient for labeling at a 1:100 dilution, although overnight incubation has shown the best results.

Controls

A number of negative controls should be performed to confirm that the section was labeled specifically. A few possibilities are listed below. Where possible, a known positive control should be included in the series to demonstrate the validity of the primary antibody and of the method.

  1. Omit the primary antibody and replace with buffer only.
  2. Replace the primary antibody with non-immune serum from the same species.
  3. Replace the primary antibody with an antibody from the same species which is non-immune or which is specific for an antigen known to be absent in the tissue.
  4. Replace the second antibody with a Bovine Serum Albumin (BSA)-Gold control at the same dilution. Use the same gold particle size.
  5. When labeling with cationic gold, incubate one section with unlabeled poly-L-Lysine (1-10mg/ml) for one hour prior to adding the conjugate.

Electron Microscope Applications (EM grade)

Efficient labeling of cells and tissues for electron microscopy depends greatly on the method of specimen preparation. Much literature continues to be published on the wide range of methods employed for tissue sections, suspended cells, cells in culture, freeze fractures, and tissue replication studies. The protocol provided in this section is a guide to optimal labeling with the indirect (two-step) method for ultrathin sections, embedded or frozen, which may be particularly helpful to those performing immunolabeling for the first time.

Mounting

Sections are best mounted on nickel or gold grids of high mesh number to improve adherence during incubations. Plastic film coating of the grids (e.g. Formvar or Butvar) is optional and depends on the stability of the sections.

Labeling Procedure

All steps may be performed at room temperature. Do not allow the sections to dry out during this procedure since this may give rise to non-specific charge attachment of the antibodies.

  1. Place the grid, section face down, on a 255l droplet of a 10% solution of heat-inactivated normal goat serum in a buffer similar to those described earlier and incubate for 10 minutes. Omit this step for Protein A and Protein G conjugates.
  2. Use a platinum wire loop or forceps to transfer the grid to the surface of a 255l droplet of appropriately diluted primary antibody. Incubate from 30 minutes to overnight (depending on dilution, temperature, etc.). Longer incubations with higher dilutions of antibody produce more specific labeling.
  3. Transfer the grid to a 255l droplet of gold conjugate diluted 1:10 to 1:100 (or greater) in PBS or Tris-buffered saline. Incubate for one hour.
  4. Transfer the grid to a series of 505l droplets of distilled water (5 x 2 minutes) to wash away unbound gold conjugates.
  5. Stain embedded sections lightly in uranyl acetate and lead citrate (optional). Frozen sections may also be stained with osmium tetroxide vapor. Wash and examine under the electron microscope.

For double labeling, the sections may be taken through steps 1-4 again before counterstaining, using the reverse side of the section. It is recommended that the first side be carbon coated by evaporation after the first incubation procedure in order to avoid contamination with the second incubation series.

Use a different size gold particle for the second round of staining.

Trouble Shooting

Problem: No label

  1. Antigen absent or destroyed due to preparative procedures. Modify procedure (e.g. fixation, cryosections, resins).
  2. Antigen present in genuinely low amounts. Use longer incubation times and/or more concentrated primary antibody.
  3. Primary antibody bad due to poor titer, age, improper storage, excessive freezing and thawing, etc. Test the antibody with positive and negative controls.
  4. pH of solutions excessively acid or alkali.
  5. Section not exposed to solutions (i.e. wrong way up) if on a plastic film.
  6. Label not visible due to heavy metal stains (e.g. for 5nm particles). Reduce counterstaining. Use higher magnification (e.g. 200,000x for 5nm; 100,000x for 10nm; 80,000x for 15nm; 50,000x for 20nm).

Problem: Excessive background staining

  1. Ionic concentration too low in solutions. Use increased salt concentration (up to 2.5%).
  2. Inadequate washing between incubations. Wash more thoroughly.
  3. Non-specific charge attraction of the antibody. Use 1% Tween in all solutions. Add ovalbumin, BSA or normal serum to approximately 1% in incubation solutions. Do not use normal serum with Protein A and Protein G. Osmium tetroxide fixation may introduce excess charge into tissue. Omit osmium or wash thoroughly after fixing.
  4. Free aldehyde groups in fixed tissue. Reduce by floating sections on a droplet of 0.5M ammonium chloride for 1 hour before incubations.
  5. Primary antibody concentration too high. Dilute by orders of magnitude.
  6. Cold conjugate concentration too high. Dilute further.
  7. Inadequately fixed tissue. Necrotic and damaged cells will also stain nonspecifically. Improve fixation conditions and use smaller tissue sections. Check the positive control.

Problem: Clustering

  1. Primary antibody aggregates present. Use fresh antisera.
  2. Natural amplification factor of the gold conjugate. Several antibody-conjugated gold particles may attach to the Fc component of the primary antibody, producing the appearance of clusters on the section. This does not occur with Protein A conjugates. Use higher dilution of gold conjugates if desired.
Literature

A number of publications deal with EM labeling protocols in detail. Some are recommended below.

  1. Polak J.M., Van Noorden S. "An introduction to immunocytochemistry: current techniques and problems." Royal Microscopical Society Handbook, 111. Oxford University Press (1984).
  2. Bullock G., Petrusz P. "Techniques in immunocytochemistry" Vols. 1,2,3. Academic Press (1983, 1984, 1985).
  3. Polak J., Varndell L.M. "Immunolabeling for electron microscopy". Elsevier Science Publishers.
  4. Polak J., Van Noorden S. "Immunocytochemistry. Modern methods and applications." J. Wright and Sons (1986).

Cationic Colloidal Gold

Introduction

Most cells of eukaryotic origin have a net negative surface charge from anionic plasma membrane components. The membrane charge distribution is thought to be important in the movement of various soluble macromolecules across cell walls. In the past, various methods have been employed to analyze the spacial distribution of anionic sites in tissues and to study the role played by surface charges in intracellular and intercellular dynamics. The newest and apparently most useful probe, cationic colloidal gold, was introduced recently. Cationic gold results from the conjugation of colloidal gold particles with poly-L-Lysine which exhibits a strong positive charge at physiological pH. A one-step incubation with these conjugates reveals subcellular sites bearing a net negative charge. The gold probes may be used at physiological pH and ionic strength. The particle sizes may be varied in order to accomplish multiple labeling. In addition, cationic gold has been tested for use in paraffin and resin embedded sections.

The method of specimen preparation may have a marked effect on the results of the experiment. The best results for electron microscopy have thus far been obtained with acrylic resins such as LR White or Lowicryl. Epoxy resins give lower labeling efficiency and increase the chance of non-specific charge interactions on the tissue surface. For light microscopy the same resins may be employed at greater thickness and these sections may be enhanced with silver. Dewaxed paraffin sections also give good results.

Labeling Procedure (also see EM or LM procedures)
  1. Wet the section with diluting buffer before applying the conjugate as this will help to avoid non-specific charge attraction.
  2. Apply 255l to 505l of diluted cationic gold to the section (1:10 to 1:100 or greater) and incubate 30 minutes to overnight at room temperature.
  3. Wash the section with diluting buffer, and then with deionized water.
  4. If using light microscopy, follow the silver enhancement procedure in the section entitled "Light Microscopy Applications."
  5. Counterstain section. For particular experiments involving electron microscopy, it may be necessary to perform tests to determine the effects of counterstaining on the avidity of the label since uranyl acetate is very acidic. For light microscopy, a light counterstain is recommended so as not to mask any of the black stain resulting from silver enhancement.
Typical results

Sections incubated with cationic colloidal gold will exhibit varying degrees of labeling in different parts of the tissue depending on the incubation conditions of the conjugate. At pH 7, intense labeling is often observed within the heterochromatin of nuclei, along plasma membranes, and within stromal components such as collagen. High labeling intensity should also be expected along components of the basal lamina within epithelial cell structures.

Trouble Shooting

Problem: Non-Specific Background

  1. Specific labeling of cell surface anionic sites may be controlled by careful adjustment of the pH during incubation, with lower pH values allowing greater specificity for these sites. The pH of the system should be reduced until background is very low but specific labeling remains high. It is advisable to perform simultaneous incubations at varying pH values in order to determine the optimal pH. Blocking agents such as bovine serum albumin may also help to minimize non-specific charge interactions.
  2. The use of osmium tetroxide, which will introduce heavy metals into tissues, is best avoided. Copper grids may also cause problems, as they can alter the charge distribution of the section.
  3. Residual aldehyde groups in formalin-fixed tissues can cause non-specific binding. Incubation with 0.1M ammonium chloride for 30 minutes will block residual aldehydes.

Literature

  1. Skutelsky E., Roth J. Cationic colloidal gold - a new probe for the detection of anionic cell surface sites by electron microscopy. J. Histochem. and Cytochem. 34:693-696 (1986).
  2. Vorbrodt A.W. Demonstration of anionic sites on the luminal and albuminal fronts of endothelial cells with poly-L-Lysine-gold complex. J. Histochem. and Cytochem. 35:1261-1266 (1987).
  3. Thurauf N., Dermietzel R., Kalweit P. Surface charges associated with fenestrated brain capillaries. J. Ultrastruct Res. 84:103-110 (1983).
  4. Beesley J., Colloidal Gold: a new perspective for cytochemical marking. Royal Microscopical Society Handbook, 17, Oxford University Press (1989).

Light Microscopy Applications (LM Grade)

The gold immunolabeling procedures for light microscopy are similar to the well established labeling methods using enzyme labeled second antibodies. A general procedure for staining smears or tissues mounted on glass slides or cells in culture, is given below. Many other procedures are available in the literature.

Labeling Procedure
  1. Dewax paraffin sections and bring to aqueous buffer (10 minutes).
  2. Place a droplet (505l) of 10% heat-inactivated normal serum (from the same host species as the gold conjugate) on the dewaxed or frozen section and incubate for 15 minutes. Do not use serum with Protein A or Protein G.
  3. Shake off the excess blocking serum and place a 505l droplet of diluted primary antibody on the section. Incubate from 30 minutes to overnight depending on dilution, temperature, etc.
  4. Wash slides extensively with dilution buffer by pipette or by immersion to remove unbound antibody.
  5. Place 505l of diluted gold conjugate (1:10 to 1:100) on the section and incubate for 1 hour.
  6. Wash extensively with deionized, distilled water to remove all unbound gold particles.
  7. Follow instructions for silver enhancement and counterstaining. The silver enhancement step will convert the invisible gold particles to an intense black reaction product.
Silver Enhancement Procedure

The following procedure is for use with BioSite Silver Enhancing Kit. Do not use any metal lab supplies in this procedure.

  1. Wash section very thoroughly with distilled or deionized water.
  2. Mix three drops each of reagent 1 (initiating solution) and reagent 2 (enhancing solution) in a clean test tube (avoid heavy metal residues).
  3. Add several drops of the mixture to the slide and monitor development of silver stain periodically under the microscope at room temperature. Do not expose slide to high intensity light for prolonged periods. Staining is typically complete in 10-20 minutes. The reaction is complete when stained areas have a brown-black stain. Stop the reaction before non-specific background appears by thorough washings in distilled water.
  4. To stop the reaction, wash slide thoroughly with water. If development is not complete, use fresh solutions and continue silver enhancement. Counterstain as desired. Mount and observe under the microscope.
Trouble Shooting

Problem: No Label

  1. Antigen absent or destroyed due to preparative procedures. Use a modified procedure (e.g. fixation, cryosections, resins).
  2. Antigen present in genuinely low amounts. Use longer incubation times and/or more concentrated primary antibody.
  3. Primary antibody poor due to poor titer, age, poor storage, excessive freezing and thawing, low avidity, wrong antibody, no antibody. Test the antibody with positive and negative controls.
  4. Buffer solutions excessively acid or alkali.
  5. Silver enhancement procedure not complete (stain is light brown or absent). Use longer incubation time with enhancement solution. Check positive controls. The same slide may be further enhanced before counterstaining.
  6. Counterstaining is masking silver stain. Use less counterstain.

Problem: Excessive background staining

  1. Ionic strength of buffers too low. Use increased salt concentration (up to 2.5%).
  2. Inadequate washing between incubations. Wash more thoroughly.
  3. Non-specific charge attraction of antibody. Use 1% detergent (e.g. Tween 20) in all solutions. Add ovalbumin, BSA or normal serum (usually goat) to approximately 1% in dilution buffers. Do not use normal serum blocking in conjunction with Protein A or Protein G.
  4. Free aldehyde groups in fixed tissue. Reduce by floating section on a droplet of 0.5M ammonium chloride for 1 hour before incubations.
  5. Primary antibody concentration too high. Dilute by orders of magnitude.
  6. Gold conjugate concentration too high. Dilute further.
  7. Inadequately fixed tissue. Necrotic and damaged cells will also stain nonspecifically. Improve fixation conditions and use smaller sections. Check the positive control.
  8. Excessive exposure to silver enhancement solution (especially where antigen concentration is low). Reduce development time.
  9. Spontaneous nucleation of silver on endogenous metals in tissue (Zn, Fe, Cd, etc.). Check negative controls.
Literature
  1. Polak J., Van Noorden S. "Immunocytochemistry: Modern Methods and Applications." J. Wright and Sons (1986).
  2. Springall, D. et al. "The potential of the immunogold silver staining method for paraffin sections." Histochemistry 81:603 (1984).


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