Single antibody procedure

Keywords Immunohistochemistry Antibody labeling Fluorescence microscopy Fluorescent immunocytochemistry Fluorescent immunohistochemistry Indirect immunocytochemistry Immunostaining 

Steps in a Single 1° Antibody Indirect Immunocytochemistry Experiment. 106 

Designing an experiment with a single 1° antibody involves many of the topics presented in the previous chapters, including reagents, types of label for antibodies, and the methods for applying antibodies. This chapter presents a step-by-step approach to designing an immunocytochemistry experiment and depends on information in the Immunocytochemistry Experimental Design Chart completed in Chapter 9. 

The example procedure presented here with rat kidney detects the antigen Ag A (Fig. 10.1a), with a mouse anti-Ag A antibody (Fig. 10.1b), and a goat anti-mouse 2° antibody labeled with a 488 fluorophore (Fig. 10.1c). 

Immunocytochemistry, DOI 10.1007/978-l-4419-1304-3 Springer Science+Business Media, EEC 2010 

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In the second category, 1° antibody, antigens are listed in separate columns so that all of the reagents associated with the E antibody can be seen. In this example, the 1° antibody to antigen Ag A is made in mouse. The dilutions of the 1° antibody for anti-Ag-A is not known, as this antibody has not be used previously, so the dilution will be determined by the Dilution Matrix when the procedure is developed in Chapter 10 (Single Antibody Procedure). 

Completing the Immunocytochemistry Experimental Design Chart guides decisions about the reagents need for the experiment. Chapter 10 (Single Antibody Procedure) presents the design strategy based on the individual steps needed for each antibody. 

Steps A-D and J-L that were covered in Chapter 10 for the single antibody procedure are the same and will not be repeated here. The discussion below will focus on the steps necessary for antibody incubations from D through I. Because both E antibodies and 2° antibodies can be combined in single incubation steps, the number of steps and the amount of reagents will be similar to those in the single 1° antibody procedure. 

Several noncompetitive assays for haptens reported so far can be regarded as variations of conventional single-antibody immunometric assays, the majority of which (the assays in Sections 3.1 and 3.2) are based on principle B1 in Fig. 4. In many cases, these methods employ an automated flow system to simplify the assay procedure and minimize the time and labor required for the analysis. 

POase added to the IgG in suitable proportions. This method gives mostly 1 1 conjugates if the molecular concentration ratio enzyme/ IgG is 4, but low yields (see Section 11.2.1). The one- and two-step GA reactions are illustrated in Fig. 11.7 and details of the two-step procedure, as described by Avrameas and Ternynck (1971), are given in Table 11.9. A feature this method shares with the single-step procedure is the extremely wasteful use of both enzyme and antibody. 

The experimental design presented in this chapter will combine the two single 1° antibody procedures into one procedure. Begin with determining the procedure for each 1° antibody separately (Chapter 10 Single 1° Antibodies). Then decide how to combine two P antibody incubations, design 2° antibody controls, and complete the final procedure. 

Single antibody experiment shows high background with labeling on some areas of the microscope slide. The procedure was as follows... 

Protocol 2 describes the procedure for generating ARM complexes. For display of a single antibody species, 1-100 ng of purified or non-purified PCR fragment is used. The amount of DNA used may depend on antibody affinity antibodies with higher affinity are more sensitively recovered and thus require less input DNA for display. 

To double label tissue with an antibody and phalloidin, follow the standard procedure for antibody labeling (see Protocol 12.2 [Labeling with a Single Antibody]) and use a fluorescent secondary antibody (step 6). Following the secondary antibody incubation, wash tissue three times in PBS/TX (10 minutes each wash). Incubate in phalloidin, rinse in PBS, and mount as outlined in steps 3-6 above. 

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