Immunogold Labeling with BioSite Colloidal Gold Reagents
General Information
Electron Microscope Applications (EM
grade)
Cationic Colloidal Gold
Light Microscopy Applications (LM Grade)
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.
- Omit the primary antibody and replace with buffer only.
- Replace the primary antibody with non-immune serum from the same
species.
- 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.
- Replace the second antibody with a Bovine Serum Albumin (BSA)-Gold
control at the same dilution. Use the same gold particle size.
- When labeling with cationic gold, incubate one section with unlabeled
poly-L-Lysine (1-10mg/ml) for one hour prior to adding the conjugate.
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.
- 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.
- 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.
- 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.
- Transfer the grid to a series of 505l droplets of distilled water
(5 x 2 minutes) to wash away unbound gold conjugates.
- 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
- Antigen absent or destroyed due to preparative procedures. Modify
procedure (e.g. fixation, cryosections, resins).
- Antigen present in genuinely low amounts. Use longer incubation
times and/or more concentrated primary antibody.
- Primary antibody bad due to poor titer, age, improper storage,
excessive freezing and thawing, etc. Test the antibody with positive
and negative controls.
- pH of solutions excessively acid or alkali.
- Section not exposed to solutions (i.e. wrong way up) if on a
plastic film.
- 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
- Ionic concentration too low in solutions. Use increased salt
concentration (up to 2.5%).
- Inadequate washing between incubations. Wash more thoroughly.
- 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.
- Free aldehyde groups in fixed tissue. Reduce by floating sections
on a droplet of 0.5M ammonium chloride for 1 hour before incubations.
- Primary antibody concentration too high. Dilute by orders of
magnitude.
- Cold conjugate concentration too high. Dilute further.
- 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
- Primary antibody aggregates present. Use fresh antisera.
- 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.
- Polak J.M., Van Noorden S. "An introduction to immunocytochemistry:
current techniques and problems." Royal Microscopical Society
Handbook, 111. Oxford University Press (1984).
- Bullock G., Petrusz P. "Techniques in immunocytochemistry" Vols.
1,2,3. Academic Press (1983, 1984, 1985).
- Polak J., Varndell L.M. "Immunolabeling for electron microscopy".
Elsevier Science Publishers.
- Polak J., Van Noorden S. "Immunocytochemistry. Modern methods
and applications." J. Wright and Sons (1986).
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)
- Wet the section with diluting buffer before applying the conjugate
as this will help to avoid non-specific charge attraction.
- 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.
- Wash the section with diluting buffer, and then with deionized
water.
- If using light microscopy, follow the silver
enhancement procedure in the section entitled "Light Microscopy
Applications."
- 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
- 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.
- 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.
- 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
- 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).
- 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).
- Thurauf N., Dermietzel R., Kalweit P. Surface charges associated
with fenestrated brain capillaries. J. Ultrastruct Res. 84:103-110
(1983).
- Beesley J., Colloidal Gold: a new perspective for cytochemical
marking. Royal Microscopical Society Handbook, 17, Oxford University
Press (1989).
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
- Dewax paraffin sections and bring to aqueous buffer (10 minutes).
- 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.
- 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.
- Wash slides extensively with dilution buffer by pipette or by
immersion to remove unbound antibody.
- Place 505l of diluted gold conjugate (1:10 to 1:100) on the section
and incubate for 1 hour.
- Wash extensively with deionized, distilled water to remove all
unbound gold particles.
- 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.
- Wash section very thoroughly with distilled or deionized water.
- Mix three drops each of reagent 1 (initiating solution) and reagent
2 (enhancing solution) in a clean test tube (avoid heavy metal
residues).
- 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.
- 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
- Antigen absent or destroyed due to preparative procedures. Use
a modified procedure (e.g. fixation, cryosections, resins).
- Antigen present in genuinely low amounts. Use longer incubation
times and/or more concentrated primary antibody.
- 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.
- Buffer solutions excessively acid or alkali.
- 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.
- Counterstaining is masking silver stain. Use less counterstain.
Problem: Excessive background staining
- Ionic strength of buffers too low. Use increased salt concentration
(up to 2.5%).
- Inadequate washing between incubations. Wash more thoroughly.
- 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.
- Free aldehyde groups in fixed tissue. Reduce by floating section
on a droplet of 0.5M ammonium chloride for 1 hour before incubations.
- Primary antibody concentration too high. Dilute by orders of
magnitude.
- Gold conjugate concentration too high. Dilute further.
- Inadequately fixed tissue. Necrotic and damaged cells will also
stain nonspecifically. Improve fixation conditions and use smaller
sections. Check the positive control.
- Excessive exposure to silver enhancement solution (especially
where antigen concentration is low). Reduce development time.
- Spontaneous nucleation of silver on endogenous metals in tissue
(Zn, Fe, Cd, etc.). Check negative controls.
Literature
- Polak J., Van Noorden S. "Immunocytochemistry: Modern Methods
and Applications." J. Wright and Sons (1986).
- Springall, D. et al. "The potential of the immunogold silver
staining method for paraffin sections." Histochemistry 81:603
(1984).
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