Protocol

Immunogold Staining of London Resin (LR) White Sections for Transmission Electron Microscopy (TEM)

This protocol was adapted from "Ultrastructural Immunochemistry," Chapter 7, in Immunohistochemistry: Methods Express (ed. Renshaw), from the Methods Express series. Scion Publishing Ltd., Oxfordshire, UK, 2006.

INTRODUCTION

In post-embedding methods of immunogold staining, the cells or tissues are fixed chemically or cryoimmobilized, dehydrated, and embedded in epoxy or acrylic resins. Thin sections (50-70 nm in thickness) are cut using an ultramicrotome with a diamond knife, using a water bath to collect the sections as they slide off the knife. The sections are stretched with solvent vapor or a heat source and collected onto either bare or plastic-coated nickel grids. The sections are then stained immunochemically with primary antibodies raised against antigens exposed on the surface of the sections. The primary antibodies are visualized by staining immunochemically with secondary antibodies raised against the species and isotype of the primary antibodies, conjugated to colloidal gold particles. The immunochemically stained sections are then contrast stained with salts of uranium (uranyl acetate) and lead (lead citrate) to reveal the ultrastructure of the cells, and are finally viewed by transmission electron microscopy (TEM). LR White was introduced as a low-toxicity alternative to epoxy resins, which frequently contained carcinogens. Unlike the simplest acrylic resins, in which monomers are polymerized to form long chains, the LR resins contain aromatic cross-linkers to improve the stability of the sections under the electron beam. LR White and Gold both have very low viscosity and readily penetrate, even into dense tissue. In this protocol, aldehyde-fixed tissue is dehydrated in ethanol, impregnated in LR White resin and polymerized under vacuum or in a nitrogen atmosphere before sectioning and immunogold staining.

RELATED INFORMATION

Ultrastructural Immunochemistry (Skepper and Powell 2008a) describes methods and considerations for the use of immunogold staining, including fixation, controls, resolution and quantification. The following protocols provide detailed procedures for immunogold staining of various sections for TEM:

Immunogold Staining of Epoxy Resin Sections for Transmission Electron Microscopy (TEM) (Skepper and Powell 2008b)

Immunogold Staining Following Freeze Substitution and Low Temperature Embedding after Chemical Fixation or after Cryoimmobilization for Transmission Electron Microscopy (TEM) (Skepper and Powell 2008c)

Immunogold Staining of Ultrathin Thawed Cryosections Sections for Transmission Electron Microscopy (TEM) (Skepper and Powell 2008d)

For more comprehensive descriptions of the range of techniques available, see Griffiths et al. (1993) and Skepper (2000).

MATERIALS

Reagents

Antibodies, primary (optimally diluted in PBSG)

Antibodies, secondary (optimally diluted in PBSG)

Use a secondary antibody raised against the species of the primary antibody and conjugated to 10- or 15-nm colloidal gold particles.

Ethanol (100%, 95%, 70% [v/v])

Formaldehyde (4%) in PIPES

Lead citrate

LR White resin (hard consistency)

Prepare a 50:50 mixture of 100% (v/v) LR White resin and 100% (v/v) ethanol.

Methanol (50%, v/v)

PBSG

Phosphate-buffered saline (PBS) (pH 7.6)

PIPES buffer (0.1 M, pH 7.4)

Potassium hydroxide

Prepare a Petri dish containing a few grains of moistened potassium hydroxide.

Sodium chloride (0.9%, w/v)

Tissue (small pieces) or cells of interest

Uranyl acetate (aqueous) (2%, w/v)

Uranyl acetate (saturated) in 50% methanol

Equipment

Dental wax (or Parafilm)

Dental wax is used as a clean hydrophobic surface on which to perform immunogold staining of thin sections mounted on TEM grids and floated on small drops of reagents.

Diamond trim tool and 45° ultradiamond knife (Diatome AG)

Incubator preset to 55°C

Microscope (transmission electron) (FEI Tecnai 120)

Nickel grids (400 mesh)

Nitrogen gas (dry) or vacuum for resin deoxygenation (see Step 7)

Polyester sheet (Melinex)

Tubes (1.5-mL microcentrifuge)

Ultramicrotome (EM UCT; Leica Microsystems)

Weighing boats (aluminum) or gelatin capsules (see Step 8)

METHOD

Tissue Embedding

1. Rinse cells or small pieces of tissue twice in 0.9% (w/v) sodium chloride.

2. Incubate in 4% formaldehyde in PIPES for 1 h at 4°C. If the cells are adherent, scrape them free from the substrate and transfer to 1.5-mL tubes.

3. Rinse cells or small pieces of tissue four times in 0.1 M PIPES buffer over a period of 20 min and twice in H₂O.

4. Incubate cells or small pieces of tissue in 2% aqueous uranyl acetate for 30 min at room temperature and rinse three times in H₂O.
See Discussion.

5. Dehydrate cells or small pieces of tissue in three changes of 70% ethanol, three changes of 95% ethanol, and three changes of 100% ethanol, all for 5 min each.

6. Incubate cells or small pieces of tissue in a 50:50 mixture of 100% LR White and 100% ethanol overnight at room temperature, and in two daily changes of 100% LR White.

7. Deoxygenate fresh resin under vacuum or by bubbling dry nitrogen gas through it for 10-20 min.

8. Place the cells or tissue in a gelatin capsule or an aluminum weighing boat.

9. Add enough resin to generate a positive meniscus and cover with a piece of Melinex sheet to exclude oxygen.
See Discussion.

10. Incubate at 55°C for 24 h to polymerize the resin.

Sectioning and Staining

All nickel grid incubations/rinses should be performed on dental wax.

11. Cut thin sections of 50-70 nm and mount onto nickel grids.

12. Incubate sections on drops of PBSG for 10 min.

13. Incubate sections on drops of optimally diluted primary antibodies in PBSG overnight.

14. Rinse sections on ten 100-µL drops of 1X PBS for 2 min on each drop.

15. Incubate sections on drops of optimally diluted species-specific secondary antibodies in PBSG (conjugated to 10- or 15-nm gold particles) at room temperature for 2 h.

16. Rinse sections in H₂O for 30-40 sec.

17. Counterstain sections by floating grids section side down on drops of uranyl acetate (saturated) in 50% methanol for 0.5-10 min at room temperature. Follow with a rinse in 50% methanol and a rinse in H₂O (Gibbons and Grimstone 1960).

18. Counterstain sections by floating grids section side down on drops of lead citrate (Reynolds 1963) for 0.5-10 min at room temperature in a Petri dish containing a few grains of moistened potassium hydroxide (to prevent lead carbonate precipitation).

19. Rinse grids extensively in H₂O and view at 80 kV in a transmission electron microscope.

DISCUSSION

LR White contains an initiator and can be polymerized by the application of heat at 48°C-50°C or by chemical catalysis at temperatures as low as -15°C to -20°C, albeit exothermically. At temperatures below -15°C, its viscosity is very high and infiltration of the resin into the tissue becomes problematic. LR Gold is less hydrophobic and can be polymerized by photoinitiation using benzoin methyl ether as a catalyst down to -25°C. At temperatures below -18°C, the initiator can spontaneously come out of solution. In procedures using LR White and Gold, aldehyde-fixed tissue is dehydrated and embedded in the acrylic resin without secondary fixation in osmium tetroxide. The tissue is polymerized in an inert atmosphere, necessary because oxygen inhibits the polymerization of these resins. Acetone is not recommended as a dehydrating agent, because it can act as a scavenger of free radicals, which can interfere with the polymerization of the resin. A convenient method for flat embedding is to place tissue in an aluminum weighing boat and exclude oxygen by dropping a piece of Melinex sheet or a Thermanox coverslip onto a positive meniscus of resin. Polymerization can be initiated chemically or photolytically at 4°C-20°C or thermally at 48°C-60°C. It is claimed that reducing the temperature during polymerization enhances antigen survival. If this is the case, in most instances the gain is likely to be marginal if the difference is a drop from 60°C to ambient temperature. Membrane preservation can be improved by bulk staining with uranyl acetate (see Fig. 1 ), before dehydration (Berryman and Rodewald 1990). The advantages of using LR White are: (i) the polymerized acrylic resin matrix is "looser" than that produced in a cured epoxy resin and (ii) the sectioning properties are different to those of epoxy resins and the antigens revealed at the surface of the section may be more accessible to the antibody molecules.

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Figure 1. Thin section through a proximal convoluted tubule of a rat kidney. The section was fixed by immersion in 2% formaldehyde and embedded in LR White after bulk staining in uranyl acetate. The basal lamina is labeled with gold particles after immunostaining for laminin. Despite the weak fixation, the outer mitochondrial membranes and cristae of mitochondria (arrows) can be clearly distinguished. Bar, 250 nm. (Reprinted with permission from Scion Publishing Ltd. © 2006.)

REFERENCES

Berryman, M.A. and Rodewald, R.D. 1990. An enhanced method for post-embedding immunocytochemical staining which preserves cell membranes. J. Histochem. Cytochem. 38: 159–170.[Abstract]

Gibbons, I.R. and Grimstone, A.V. 1960. On flagellar structure in certain flagellates. J. Biophys. Biochem. Cytol. 7: 697–716.[Medline]

Griffiths, G., Burke, B., and Lucocq, J. 1993, Fine structure immunocytochemistry.

Reynolds, E.S. 1963. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17: 208–212.[Free Full Text]

Skepper, J.N. 2000. Immunocytochemical strategies for electron microscopy: Choice or compromise. J. Microsc. 199: 1–36.[Medline]

Skepper, J.N. and Powell, J.M. 2008a. Ultrastructural immunochemistry. CSH Protocols (this issue) doi: 10.1101/pdb.top47.[Abstract/Free Full Text]

Skepper, J.N. and Powell, J.M. 2008b. Immunogold staining of epoxy resin sections for transmission electron microscopy (TEM). CSH Protocols (this issue) doi: 10.1101/pdb.prot5015.[Abstract/Free Full Text]

Skepper, J.N. and Powell, J.M. 2008c. Immunogold staining following freeze substitution and low temperature embedding after chemical fixation or after cryoimmobilization for transmission electron microscopy (TEM). CSH Protocols (this issue) doi: 10.1101/pdb.prot5017.[Abstract/Free Full Text]

Skepper, J.N. and Powell, J.M. 2008d. Immunogold staining of ultrathin thawed cryosections for transmission electron microscopy (TEM). CSH Protocols (this issue) doi: 10.1101/pdb.prot5018.[Abstract/Free Full Text]