Experimental design chart 1 antibody

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. 

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. 

In planning experiments, it is important to look at the entire experiment and know that all of the reagents and conditions have been considered. Table 10.1 is the Experimental Design Chart for a single antibody experiment. The most common use for single antibody experiments is to test a new 1° antibody and to perform the Dilution Matrix described later. Before planning the details needed to perform each of the steps in the experiment, this chart must be completed. 

Experimental Design Chart for Block-Between Method. Design the 2° Antibody Control for the Same Species... 

A new Experimental Design Chart must be used for this Indirect Immunocytochemistry Block-Between (Table 12.1), because it differs from the chart used previously. Here, the experiment uses cultured cells on coverslips and two 1° antibodies made in mouse, mouse anti-Ag A and mouse anti-Ag B. A new row on the Chart is needed, 4. Block-between, to list two new reagents used to block-between the two 1° antibodies. Normal mouse serum is used at 1 20, which is sufficient to block in most cases. The anti-mouse Fab molecule is used at 20 p.g/ml. Incubations for each of these steps is for 1 h with six rinses after each incubation. Note these blocking incubations must be performed in the order of normal mouse serum first and anti-mouse Fab second. 

To plan an experiment, refer to the Experimental Design Chart for Indirect Immunocytochemistry Zenon with mouse anti-Ag A and a mouse anti-Ag B 1° antibodies (Table 12.3). In the chart Section 2. 1° Antibodies include the isotype of each mouse 1° antibodies. Use this information in selecting a Fab-labeling reagent for the 1° antibodies. Use chart Section 3. 1° Antibody Labeling, to help select the... 

Experimental Design Chart - a guide to the selection of antibodies, incubation conditions, and solutions in planning an immunocytochemical experiment. 

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