Immunostaining protocol

Further dilute (if required) the resultant primary antibody Fab fragment complexes to optimal working concentration (usually about 1 5 pg/ml) in staining buffer containing 10% normal serum and then apply to the sample for 30 60 min at room temperature and proceed further with your standard immunostaining protocol. 

Before the beginning of the immunostaining protocol, slides should be put in a Coplin jar filled with an antigen retrieval solution of choice and heated in a commercial microwave oven operating at a frequency of 2.45 GHz and 600 W power setting. After two heating cycles of 5 min each, slides should be allowed to cool at room temperature and thoroughly washed in PBS. 

Proceed immediately with the immunostaining protocol (see Chapters 24-27,29, 43-45) (see Notes 12-16). 

Proceed immediately with the immunostaining protocol (see Notes 12-16). 

Following rinsing in PBS, the sections are treated with free biotin solution (2.5 mg in 0.1 mM PBS) for 4 min to saturate the remaining binding sites of the egg white avidin. The sections are thoroughly rinsed with tromethamine-based buffer or PBS, followed by the following sequential immunostaining protocol biotinylated secondary antibody,... 

Both negative and positive tissue controls should be processed using the same fixation, embedding, mounting, drying, epitope retrieval and immunostaining protocols as the patient tissue. 

Shidham VB, Komorowski R, Macias V, et al. Optimization of an immunostaining protocol for the rapid intraoperative evaluation of melanoma sentinel lymph node imprint smears with the MCW melanoma cocktail . Cytojournal. 2004 1 2. 

Test antibodies mouse monoclonal anti-Ki67/Mibl (1 150), anti-bcl2/124 (1 100), anti-CD35/To5 (1 100), and rabbit polyclonal anti-CD3 (all from Dako). Incubation 60 min at room temperature. (For immunostaining protocols, see Chapters 25, 26, 28, and 29)). 

Below are four fixation/immunostaining protocols. Each of these protocols proved to be suitable for the immunolocalization of one or more proteins along mitotic chromosomes. 

Immunocytochemistry and Kelated Techniques is a collection of protocols for the immunocytochemical analysis of neurons and neural networks where emphasis is given not only to the immunostaining protocol per se but also to its possible ameliorations in qualitative and quantitative terms, and to the possibility of employing immunocytochemical labeling as a part of a more comprehensive approach to understand the structure and function of the brain. 

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