Experimental design chart

The chart is divided on the left into six categories that should be followed from the top down to complete the chart for each experiment. The Appendix contains chart templates for various methods of antibody detection presented in this book. The second column from the left lists parameters within each category where specific information is needed. The boxes surrounded by dark lines on the right side of the chart (filled in this example) indicate two types of information needed. The column with the heading, Conditions, collects information about the procedure independently of the antibodies to be used. The last column. Antigen No. 1, asks for information specific for each antibody to be used. Most of the experiments scientists perform use at least two antibodies and the chart allows for one antibody additional columns can be added for more antibodies. 

The first category (Table 9.2), (1) Sample, collects the conditions of sample preparation. In the example here, kidney from a rat will be used following trans-cardiac perfusion. The tissue blocks will be infiltrated with 20% sucrose overnight, frozen in isopentane, and sectioned on a cryostat. For processing, the sections will be placed on a microscope slide. To calculate how much incubation and rinse solutions are needed, the size of the incubation chamber is listed. In the example, 250 p,l will just cover the tissue and can be used for each step. 

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). 

The third category, 2° antibody, summarizes 2° antibody and label information. This information allows the scientist to check the species of the 2° antibodies so that no cross-reactive antibodies will be used. In addition, the information about the fluorophores lists the excitation and emission wavelengths to show that different nonoverlapping fluorophores will be used. 

The fourth category. Incubation Solutions, includes information on the species of the normal blocking serum and the detergent. 

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Each chapter follows the order of these procedure steps and explains different options. The goal is to give enough information to design a procedure for a particular experimental need. In Chapter 9, Decision Method and Label, an Experimental Design Chart is presented that guides the decisions on reagents for specific experiments. 

Table 9.2 Experimental design chart Experimental design chart Indirect immunocytochemistry...

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... 

Experimental Design Chart for the Same Species with Zenon. 130... 

Experimental Design Chart for Block-Between Method... 

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... 

With the immunocytochemistry protocol completed, the tissue or cell preparation is now ready to be examined with a microscope. The type of microscope and the location of the microscope were determined in Chapter 9 when the Immunocytochemistry Experimental Design Chart and Section 6, Microscopy, were completed. 

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

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