ABSTRACT
This chapter provides an overview of methods for applying immunolabeling to the realm of electron microscopy (EM). A wide range of xation, embedment, sectioning, and staining techniques are available for ultrastructural immunostaining to which the limited space available allows only a brief introduction. For more detailed
12.1 Introduction .................................................................................................. 343 12.2 Antibodies ..................................................................................................... 345
12.2.1 Primary Antibodies .......................................................................... 345 12.2.2 Troubleshooting and Storage ............................................................346 12.2.3 Secondary Reagents ..........................................................................346 12.2.4 Titration of Reagents......................................................................... 347 12.2.5 Background Staining ........................................................................348 12.2.6 Controls ............................................................................................. 349
12.3 Labels ............................................................................................................ 350 12.3.1 Colloidal Gold................................................................................... 350 12.3.2 Other Electron-Dense Labels ........................................................... 352 12.3.3 Pitfalls and Hints for Visualizing Heavy Metal Labels.................... 353 12.3.4 Multiple-Labeling Applications ........................................................ 353 12.3.5 Quantication of Labeling ................................................................ 354
12.4 Tissue Substrates for Labeling ...................................................................... 355 12.4.1 Fixation ............................................................................................. 355 12.4.2 Preparation for Ultramicrotomy ....................................................... 356
12.5 Labeling Methods ......................................................................................... 356 12.5.1 Labeling of Frozen Sections ............................................................. 356 12.5.2 Post-Embedment Labeling of Nonfrozen Tissues ............................ 359 12.5.3 Pre-Embedment Labeling .................................................................360 12.5.4 Particulate Specimens....................................................................... 362 12.5.5 Replica Labeling ............................................................................... 363
12.6 Correlative Immunomicroscopy ...................................................................364 References ..............................................................................................................364
