Direct &indirect immunofluorescence - أطلس مختبرات العرب

Immunofluorescence detects antigen in situ. A section is cut on a cryostat from a deep-frozen tissue block. This ensures that labile antigens are not damaged by fixatives.

Direct: the test solution of fluoresceinated antibody is applied to the section in a drop, incubated and washed off. Any bound antibody is then revealed under the microscope; UV light is directed onto the section through the objective, thus the field is dark and areas with bound fluorescent antibody fluoresce green. The pattern of fluorescence is characteristic for each tissue antigen.

Indirect: antibody applied to the section as a solution is visualized using fluoresceinated anti-immunoglobulin.

Indirect complement amplified: this is an elaboration of the indirect method for the detection of complement-fixing antibody (see Fig. 27.7). In the second step fresh complement is added which becomes fixed around the site of antibody binding. Due to the amplification steps in the classical complement pathway (see Chapter 3) one antibody molecule can cause many C3b molecules to bind to the section; these are then visualized with fluoresceinated anti-C3.