Indirect immunocytochemistry

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

Here then is how it all works together. Immunocytochemistry takes tissue sections or culture cells and incubates them with antibodies. The experimental needs determine the exact order of antibodies incubations and the specific labeling of the antibodies. The general steps in a single primary (1°) antibody indirect immunocytochemistry experiment include the following ... 

Fig. 7.2 Indirect immunocytochemistry. An unlabeled 1° antibody binds to the antigen and a labeled 2° antibody binds to the 1° antibody. The labeled 2° antibody is made in a different species of animals against the IgG species of the 1° antibody. A variety of labels can be used from fluorescence to enzymes...

A major advantage to the indirect method is that each labeled 2° will attach to all 1° antibodies from one species (e.g., goat anti-rabbit IgG labeled with 488 flu-orophore works for all rabbit 1° antibodies). This is the method of choice today because so many different antibodies are used in biomedical research. Indirect immunocytochemistry detects proteins in cells with the high detection sensitivity, good flexibility of reagents, and the fewest steps. 

Offers modest increase in sensitivity over indirect immunocytochemistry with fluorescent-labeled 2°. 

One advantage of indirect avidin-biotin method is an amplification of the detection system. For example, after biotin conjugated 2° antibody, avidin is incubated, binding to the available biotins. After the remaining free avidin is rinsed off, a biotin conjugated to 488 fluorophore is added and it binds to the remaining sites on the avidin. With this method, amplification occurs because any avidin can be attached to multiple biotins conjugated with either fluorescent or enzyme. This method requires two additional incubation steps of the indirect immunocytochemistry with fluorescent-labeled 2°. 

Requires two additional incubation steps over indirect immunocytochemistry. 

ABC greatly improves detection sensitivity over indirect immunocytochemistry. 

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

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

Steps in a two 1° antibody different species indirect immunocytochemistry experiment. 

When an experiment uses multiple 1° antibodies derived from the same species, perform the incubations for the first 1° and 2° antibodies, block the remaining antibody sites, and then perform the incubations for the second 1° and 2° antibodies. This procedure consists of a series of two single U antibody indirect immunocytochemistry experiments with extensive blocking between them. The key element is the blocking steps between. In this example, cultures are incubated with the first 1° antibody set, mouse anti-Ag A and 2° antibody goat anti-mouse labeled with 488 fluorophore (Fig. 12.1a), followed by steps that block the remaining antibody sites (Fig. 12.1b, c). The incubations with the second 1° antibody set are with mouse anti-Ag B antibody and 2° antibody goat anti-mouse... 

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. 

Table 12.2 Controls for indirect immunocytochemistry multiple antibodies with block between...

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... 

Direct immunocytochemistry Indirect immunocytochemistry one sample Indirect immunocytochemistry two sample Indirect immunocytochemistry block-between Indirect immunocytochemistry Zenon ... 

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