Immuno-gold labelling

Immunogold labelling of biological specimens enables the precise localization and quantification of proteins at the electron microscopy level of resolution. Ultrathin sections are labelled with antibodies specific to the target antigen and subsequently labelled with gold particles. Gold particles of varying diameters can be utilized to study two or more proteins simultaneously.

The EM Unit provides sample preparation for post-embedding immunogold labelling of samples. The investigator is responsible for supplying the primary and secondary antibodies and labelling the provided sections (immunolabeling at the fluorescent light microscopy level before attempting it at the EM level is recommended).

Sample preparation follows a similar protocol to conventional methods with certain adjustments to preserve protein antigenicity and enhance labelling efficiency. Conventional protocols for preparing samples for immunogold labelling includes the following steps:

Primary fixation with aldehydes (proteins): During this step, proteins and other cell molecules are crosslinked using formaldehyde and/or glutaraldehyde. The specimen should be dissected to a maximum thickness of 1 mm in at least one direction and fixed through immersion (using buffered 2% paraformaldehyde, 0.5% glutaraldehyde).

Quenching: A quenching agent is employed to neutralize the charged aldehyde groups, which may otherwise bind non-specifically to antibodies (using 0.1% sodium borohydride / 0.02 M glycine).

Dehydration series with solvent (ethanol): The fixed specimen undergoes dehydration through a series of ethanol solutions. The solvent concentration is gradually increased to gently remove water without causing artefacts, particularly shrinkage.

Resin infiltration and embedding: Following dehydration, the solvent is replaced with a gradually increasing concentration of liquid resin (LR White). The specimen is placed in a gelatine capsule filled with liquid resin and cured into a solid block. Once completed, the sample can be stored indefinitely.

Sectioning and mounting sections on specimen grids: A specimen embedded in hardened resin can be thinly sectioned and mounted on nickel specimen grids.

Etching (optional): In certain cases, sections may undergo deplasticization to facilitate epitope retrieval and enhance labelling efficiency (using 20% H2O2).

Blocking: To prevent nonspecific binding of antibodies, sections must be blocked using inert proteins as competitors (using 5% BSA/Tris buffer saline TBS or BSA/PBS buffer).

Primary labelling: The sections are labelled with antibodies specific to the target antigen (using the primary antibody in an appropriate buffer, e.g., 1% BSA/TBS). Note: Longer incubation times and higher antibody dilutions result in more specific labelling.

Secondary labelling: The primary antibody is labelled with secondary antibodies conjugated to a colloidal gold particle (using the secondary antibody conjugated with gold in an appropriate buffer, e.g., 1% BSA/TBS). Note: Gold is utilized for its high electron density, which increases electron scatter, leading to high contrast "dark spots."

Contrasting: Biological specimens are naturally not very electron-opaque due to their composition of low atomic number atoms, which allows the electron beam to pass through easily. To enhance sample contrast, the sections can be post-stained with lead citrate and uranyl acetate, which bind to cell components and scatter the incident beam.