Incubation of reagents

Incubation of reagents on the slides for the proper length of time, followed by appropriate rinsing. 

Figure 18.14 NHS-SS-PEG4-biotin can be used to label a primary antibody molecule that has specificity for a protein or interest. Incubation of the biotinylated antibody with a sample, such as a cell lysate, allows the antibody to bind to its target. Capture of the antibody-antigen complex on an immobilized streptavidin reagent effectively isolates the targeted protein from the other proteins in the sample. The disulfide linkage in the spacer arm of the biotin tag permits elution of the immune complex from the streptavidin support using DTT and without using the strong denaturing condition typically required to break the streptavidin-biotin interaction.

Prepare the ABC reagent according to the manufacturer s instructions. For Vector Laboratories Vectastain Elite kit, add 2 drops of reagent A to 5 mL of PBS, then add 2 drops of reagent B. Mix and incubate for 30 min at room temperature. Additional volume may be prepared keeping the ratio of reagents constant. Repeat step 6. 

The advantages of the Leica/Jung instrument are that incubation temperatures can he elevated for shorter runs, humidity is controlled, and the amount of antibody applied to a slide can be customized for the amount of tissue present. There was, however, a low level of reagent carryover similar to that observed with the Cadenza. See Subheading 2.2.3. for recommendations for detection and prevention of this potential problem. 

An alternative method of overcoming the time delay of mixing is to use a relaxation method. An equilibrium mixture of reagents is preincubated and the equilibrium is perturbed by an external influence. The rate of return, or relaxation, to equilibrium is then measured. The most common procedure for this is temperature jump (Figure 4.6).13 A solution is incubated in an absorbance or fluorescence cell and its temperature is raised through 5 to 10°C in less than a microsecond by the discharge of a capacitor (or, in more recent developments, in 10 to 100 ns by the discharge of an infrared laser). If the equilibrium involves an... 

Wipe excess liquid from the area around the sections, and apply sufficient 5% heat-inactivated normal serum from the second antibody species to cover each entire section (15 min). If protein A-gold probes are used, flood the sections with BSA-PBS rather than normal serum. It is important to keep the sections covered and in a humid environment throughout all incubations, preferably using a purpose-made humidity chamber. Depending on the size of the section, about 50-200 uL of reagent is sufficient to cover each preparation... 

For focused libraries, where one amine is converted to ca. 80 analogs, 80 polypropylene fritted vessels or wells of a 96-deepwell plate are charged with 80 different TFP reagents. The focused library is obtained following incubation of the dispensed resins with one amine and workup as described above. Analytical data are collected on all the members of the library. 

Butz, W. R., Clark, C. C., and Miller, R. T. 1994. Improved manual immunohistochemistry employing orbital mixing of reagents during incubations. Appl. Immunohistochem. 2 65-67. 

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