Department of Pathology: Confocal Facility

Protocols:

The following protocol documents are available in Microsoft Word format.

Immunofluorescence Paraffin Sections (TSA Method)
This protocol will enable you to label three proteins by immunofluorescent staining using formalin fixed, paraffin embedded tissue when two of your antibodies are made in the same species of animal (i.e. rabbit) or if one of your antigens is relatively rare. By amplifying the first antibody with tyramide at a dilution that could not be visualized without amplifcation, a second antibody produced in the same species can be used subsequently.

Cell Staining
This protocol can be used for double immunofluorescent labeling using tyramide amplification on cells grown on coverslips. Tyramide amplification will allow detection of rare proteins or can be used in cases when the two antibodies of interest are made in the same species. The first antibody (the one amplified by tyramide) should be used at a dilution in which no signal could be detected without amplification.

Cell Staining-Strep Amplification: Double Labeling
For stoichiometric amplification of your first antibody (4 fold), we use biotin conjugated secondary antibodies followed by streptavidin conjugated fluors. A second antibody without amplification can follow the first antibody.

Cell Staining-No Amplification: Double Labeling
For immunofluorescent labeling of 2 proteins without amplification, please use this protocol.

Cell Staining-Indirect TSA Amplification: Triple Labeling
For triple immunofluorescent labeling of cells grown on coverslips using the tyramide amplification of the first antibody. Amplification is used if your protein is in low abundance or if the antibody to your protein is made in the same species as another antibody you wish to label with simultaneously. A fourth dye to label cell nuclei is also listed.