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

Jeremy N. Skepper and Janet M. Powell

This protocol was adapted from “Ultrastructural Immunochemistry,” Chapter 7, inImmunohistochemistry: Methods Express (ed. Renshaw), from the Methods Express series. ScionPublishing Ltd., Oxfordshire, UK, 2006.

INTRODUCTION

In post-embedding methods of immunogold staining, the cells or tissues are fixed chemically orcryoimmobilized, dehydrated, and embedded in epoxy or acrylic resins. Thin sections (50-70 nm inthickness) are cut using an ultramicrotome with a diamond knife, using a water bath to collect thesections as they slide off the knife. The sections are stretched with solvent vapor or a heat source andcollected onto either bare or plastic-coated nickel grids. The sections are then stained immuno-chemically with primary antibodies raised against antigens exposed on the surface of the sections. Theprimary antibodies are visualized by staining immunochemically with secondary antibodies raisedagainst the species and isotype of the primary antibodies, conjugated to colloidal gold particles. Theimmunochemically 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 transmissionelectron 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 arepolymerized to form long chains, the LR resins contain aromatic cross-linkers to improve the stabilityof the sections under the electron beam. LR White and Gold both have very low viscosity and readilypenetrate, 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 beforesectioning and immunogold staining.

RELATED INFORMATION

Ultrastructural Immunochemistry (Skepper and Powell 2008a) describes methods and considera-tions for the use of immunogold staining, including fixation, controls, resolution and quantification.The following protocols provide detailed procedures for immunogold staining of various sectionsfor 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 afterChemical Fixation or after Cryoimmobilization for Transmission Electron Microscopy (TEM)(Skepper and Powell 2008c)

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

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

© 2008 Cold Spring Harbor Laboratory Press 1 Vol. 3, Issue 6, June 2008

Please cite as: CSH Protocols; 2008; doi:10.1101/pdb.prot5016 www.cshprotocols.org

Protocol

Cold Spring Harbor Laboratory Press on November 20, 2020 - Published by http://cshprotocols.cshlp.org/Downloaded from

www.cshprotocols.org 2 CSH Protocols

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 freefrom 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 andtwice in H

2O.

4. Incubate cells or small pieces of tissue in 2% aqueous uranyl acetate for 30 min at room tem-perature and rinse three times in H

2O.

See Discussion.

MATERIALS

CAUTIONS AND RECIPES: Please see Appendices for appropriate handling of materials marked with <!>, andrecipes for reagents marked with <R>.

Reagents

<R>Antibodies, primary (optimally diluted in PBSG)<R>Antibodies, secondary (optimally diluted in PBSG)

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

Ethanol (100%, 95%, 70% [v/v])<R>Formaldehyde (4%) in PIPES<!>Lead citrateLR 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)<R>PBSG<R>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)<R>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 sectionsmounted on TEM grids and floated on small drops of reagents.

Diamond trim tool and 45° ultradiamond knife (Diatome AG)Incubator preset to 55°CMicroscope (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)

Cold Spring Harbor Laboratory Press on November 20, 2020 - Published by http://cshprotocols.cshlp.org/Downloaded from

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% ethanolovernight 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 toexclude 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) in50% methanol for 0.5-10 min at room temperature. Follow with a rinse in 50% methanol and arinse in H

2O (Gibbons and Grimstone 1960).

18. Counterstain sections by floating grids section side down on drops of lead citrate (Reynolds1963) for 0.5-10 min at room temperature in a Petri dish containing a few grains of moistenedpotassium 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 bychemical catalysis at temperatures as low as -15°C to -20°C, albeit exothermically. At temperaturesbelow -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 asa catalyst down to -25°C. At temperatures below -18°C, the initiator can spontaneously come out ofsolution. In procedures using LR White and Gold, aldehyde-fixed tissue is dehydrated and embeddedin the acrylic resin without secondary fixation in osmium tetroxide. The tissue is polymerized in aninert atmosphere, necessary because oxygen inhibits the polymerization of these resins. Acetone is notrecommended as a dehydrating agent, because it can act as a scavenger of free radicals, which caninterfere with the polymerization of the resin. A convenient method for flat embedding is to place tis-sue in an aluminum weighing boat and exclude oxygen by dropping a piece of Melinex sheet or aThermanox coverslip onto a positive meniscus of resin. Polymerization can be initiated chemically orphotolytically at 4°C-20°C or thermally at 48°C-60°C. It is claimed that reducing the temperature dur-ing polymerization enhances antigen survival. If this is the case, in most instances the gain is likely tobe marginal if the difference is a drop from 60°C to ambient temperature. Membrane preservationcan be improved by bulk staining with uranyl acetate (see Fig. 1), before dehydration (Berryman andRodewald 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 tothose of epoxy resins and the antigens revealed at the surface of the section may be more accessibleto the antibody molecules.

www.cshprotocols.org 3 CSH Protocols

Cold Spring Harbor Laboratory Press on November 20, 2020 - Published by http://cshprotocols.cshlp.org/Downloaded from

Berryman, M.A. and Rodewald, R.D. 1990. An enhanced method forpost-embedding immunocytochemical staining which preservescell membranes. J. Histochem. Cytochem. 38: 159–170.

Gibbons, I.R. and Grimstone, A.V. 1960. On flagellar structure in cer-tain flagellates. J. Biophys. Biochem. Cytol. 7: 697–716.

Griffiths, G., Burke, B., and Lucocq, J. 1993. Fine structure immunocy-tochemistry. Springer-Verlag, Heidelberg, Germany.

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.

Skepper, J.N. 2000. Immunocytochemical strategies for electronmicroscopy: Choice or compromise. J. Microsc. 199: 1–36.

Skepper, J.N. and Powell, J.M. 2008a. Ultrastructural immunochem-

istry. CSH Protocols (this issue) doi: 10.1101/pdb.top47.Skepper, J.N. and Powell, J.M. 2008b. Immunogold staining of epoxy

resin sections for transmission electron microscopy (TEM). CSHProtocols (this issue) doi: 10.1101/pdb.prot5015.

Skepper, J.N. and Powell, J.M. 2008c. Immunogold staining follow-ing freeze substitution and low temperature embedding afterchemical fixation or after cryoimmobilization for transmissionelectron microscopy (TEM). CSH Protocols (this issue) doi:10.1101/pdb.prot5017.

Skepper, J.N. and Powell, J.M. 2008d. Immunogold staining of ultra-thin thawed cryosections for transmission electron microscopy(TEM). CSH Protocols (this issue) doi: 10.1101/pdb.prot5018.

www.cshprotocols.org 4 CSH Protocols

REFERENCES

FIGURE 1. Thin section through a proximal convolutedtubule of a rat kidney. The section was fixed by immer-sion in 2% formaldehyde and embedded in LR Whiteafter bulk staining in uranyl acetate. The basal lamina islabeled with gold particles after immunostaining forlaminin. Despite the weak fixation, the outer mitochon-drial membranes and cristae of mitochondria (arrows)can be clearly distinguished. Bar, 250 nm. (Reprintedwith permission from Scion Publishing Ltd. © 2006.)

Cold Spring Harbor Laboratory Press on November 20, 2020 - Published by http://cshprotocols.cshlp.org/Downloaded from

doi: 10.1101/pdb.prot5016Cold Spring Harb Protoc;  Jeremy N. Skepper and Janet M. Powell Electron Microscopy (TEM)Immunogold Staining of London Resin (LR) White Sections for Transmission

ServiceEmail Alerting click here.Receive free email alerts when new articles cite this article -

CategoriesSubject Cold Spring Harbor Protocols.Browse articles on similar topics from

(84 articles)Visualization of Organelles (127 articles)Visualization of Gene Expression

(516 articles)Visualization (35 articles)Phenotypic Analysis (333 articles)Labeling for Imaging (44 articles)Immunostaining Cells

(119 articles)Immunostaining (119 articles)Immunology, general

(40 articles)Immunoimaging (81 articles)Immunohistochemistry

(575 articles)Imaging/Microscopy, general (43 articles)Electron Microscopy

(521 articles)Cell Imaging (1352 articles)Cell Biology, general

(257 articles)Antibodies, general

http://cshprotocols.cshlp.org/subscriptions go to: Cold Spring Harbor Protocols To subscribe to

Cold Spring Harbor Laboratory Press on November 20, 2020 - Published by http://cshprotocols.cshlp.org/Downloaded from