Procedures for ADC Immunoblotting and Immunolocalization for Transmission Electron Microscopy During Organogenic...

Immunolocalization for transmission electron microscopy is a powerful technique to identify subcellular localization of proteins. This can be combined with molecular and physiological data in order to have a complete overview of protein function. However, optimal sample preservation is required to avoid artefacts. When using chemically fixed samples, the progressive lowering of temperature (PLT) technique is a convenient procedure to dehydrate and embed samples at low temperature, thereby preserving the antigenicity of the proteins to be detected. Despite the advantages of immunogold labelling, it is a time-consuming cell biology technique. Therefore, the quality and specificity of the antibody should be previously checked by western blot. This approach also enables to identify changes in the amount of protein under study throughout development or in response to stress conditions.