Immunogold labeling (chemical fixation)
The samples are fixed with 4% paraformaldehyde and 0.01% glutaraldehyde in 0.1 M buffer. Fixative can be collected at our laboratory prior experiment. After fixation the samples are dehydrated in increasing concentrations of ethanol. After dehydration the samples are embedded in resin (LR White or Lowicryl) and polymerized by heat or UV-light.
It is possible to do pre-immunogold labeling, in which the antibodies are added befor resin embedding or post-immunogold labeling, in which the antibodies are added after resin embedding, i.e. directly on ultrathin sections. It is also possible to combine immunogold labeling with negative staining.
We can provide with gold-conjugated secondary antibodies (anti-mouse and anti-rabbit). If small/ultrasmall gold particles are used (<2nm), we can do silver enhancment to increase the size and visability of the gold particles.
Note 1: To increase the antigenicity, Osmium Tetraoxide and Epon resin are normally omittet. This will lead to an impairment of the ultrastructure.
Note 2: We encourage to first test the primary antibodies with another technique, e.g. immunofluorescence before performing immunogold labeling.