We have introduced a new policy for EM  immunogold labeling projects.

To avoid disappointment with the outcome of an immunolabeling project we now REQUIRE the following prerequisites from investigators:

1) A gel showing antibody specificity - a single band should be present - note some commercial vendors show a gel run against the specific protein only. The gel needs to be run against a cell lysate or similar

2) Successful immunofluorescence labeling with fixatives compatible with electron microscopy. We will supply suitable fixatives for testing.

3) The protocol used for immunofluorescence - we need to know successful primary antibody concentrations - be aware the electron microscope immunolabeling usually requires at least a 100x increase in concentration over immunofluorescence.

We have a number of approaches that we can use for immunogold labeling. It is not possible to predict in advance which will be the most successful method - every antigen is different! High quality structure preservation and preservation of antigens are often mutually incompatible.

The simplest and most convenient method is to fix in a paraformaldehyde/glutaraldehyde mixture followed by gentle dehydration and embedding in acrylic resin (LR White)

When this is not successful we can try a similar fixation but use sucrose cryoprotection followed by rapid freezing and cryosectioning (Tokoyasu technique).

Finally we can use high pressure freezing and freeze substitution. This is a good method but labour intensive and time consuming.

We have a robot to handle the immunolabeling. This means we can reliably and reproducibly handle a number of samples and antibody concentrations in a single run.