Vector Labs

Immunofluorescence Staining Methods
Immunofluorescence staining methods can also be used successfully for labeling multiple antigens in the same preparation. These methods are especially useful for co-localization of antigens in the same compartment of a cell, and in this regard offer a distinct advantage over enzyme-based detection systems.  
Traditionally, double fluorescent labeling has been achieved by using fluorescently conjugated secondaries against primary antibodies from different species. Although this technique in general tends to be less sensitive than enzymatic staining, the sensitivity of the fluorescent stain can be increased by using a (strept)avidin-biotin system for amplification of the label. Many of the principles regarding multiple antigen labeling will apply to both immunofluorescent and enzymatic methods (see section on “Immuno-enzymatic Staining Methods”). For best results, sequential staining of each primary antibody is recommended. As in enzymatic applications, the order of labeling, appropriate controls, and additional blocking steps may be important to obtain optimal results. In contrast to enzymatic staining, special consideration must be given to the species of the primary antibodies to be used. For fluorescent applications, the two primary antibodies usually should be from different species, otherwise, artifactual co-staining of antigens may result. However, the use of two mouse primary antibodies is the exception. See section on “Multiple Immunofluorescent Labeling Using Two or More Mouse Monoclonal Primary Antibodies”.  
In addition, if the (strept)avidin biotin system is used for the visualization of both antigens, a (strept)avidin-biotin block MUST be used before the second primary antibody step.  
Following labeling it is important to preserve the intensity of the fluorescent signal, as some fluorescent products are prone to rapid fading when viewed under the microscope. In most instances this is easily accomplished by coverslipping the preparation with an anti-fade, anti-photobleaching agent such as VECTASHIELD® mounting media. The use of VECTASHIELD® also allows the preparation to be stored for extended periods without significant loss of intensity.  
Autofluorescence of the tissue may obscure staining depending on the type of tissue, the fixation method, and the microscope filter used for visualization. This should be evaluated before staining, and autofluoresence quenched, if necessary, using appropriate methods.  
The following protocols are for frozen tissue sections. These protocols can be adapted for other tissue preparations.  
Protocol: Double Immunofluorescent Labeling Using Primary Antibodies from Different Species  
Protocol: Multiple Immunofluorescent Labeling Using Two or More Mouse Monoclonal Primary Antibodies  
Colon (frozen) – Double label • Multi-Cytokeratin (m), M.O.M.™ Fluorescein Kit (green). • Desmin (m), M.O.M.™ Basic Kit, Texas Red® Avidin DCS (red).  
Tonsil (frozen) – Double label • Ki67 (m), M.O.M.™ Fluorescein Kit (green). • Pan-Cytokeratin (m), M.O.M.™ Basic Kit, Texas Red® Avidin DCS (red).  
Small Bowel (frozen) – Double label • PGP9.5 (m), M.O.M.™ Basic Kit, VECTASTAIN® ABC-AP Standard Kit, Vector® Red AP substrate (red). • Desmin (m), M.O.M.™ Fluorescein Kit (green).