Heat-Assisted Immunofluorescence Staining of Tissue Sections

MICROWAVE HEAT-ASSISTED IMMUNOFLUORESCENCE STAINING OF TISSUE SECTIONS 

Frozen sections, as well as sections of formaldehyde-fixed and paraffin-embedded tissues, can be used for direct or indirect immunofluorescence staining after antigen retrieval using microwave heating. Although frozen sections yield higher sensitivity than that obtained with paraffin sections, the latter approach is necessary for retrospective studies of archival specimens. Moreover, fresh tissue is not always available. 

Sections (4 xm thick) of formalin-fixed and paraffin-embedded tissues are deposited on a coated slide, deparaffinized, rehydrated, and rinsed in PBS. They are placed in sodium citrate buffer-containing plastic jars and heated twice for 5 min each in a microwave oven. Following cooling at room temperature for 20 min, the sections are treated with 0.3% trypsin. They are washed in PBS, blocked with normal serum for 10 min, and then overlaid with fluorescein-conjugated rabbit antibodies to human IgG at 1 20 dilution in PBS for 45 min at room temperature. 

The sections are washed in PBS for 5 min and incubated for 30 min in fluorescein isothiocyanate (FICT)-labeled swine antirabbit immunoglobulin conjugate at 1 20 dilution in PBS. After being washed in PBS for 5 min, the sections are mounted with an aqueous mounting medium. The sections are observed under an epiilluminating fluorescent microscope. The negative control is not incubated with the primary antibody, and a frozen section from a patient with the known condition is used as the positive control. 

Double immunofluorescence labeling in conjunction with microwave heating can be used to visualize two markers at the same cellular location in routine formalin-fixed and paraffin-embedded tissue sections (Mason et al 2000). The primary antibodies are either monoclonal antibodies of differing isotype/subclass or antibodies from different species. Labeling is visualized on a conventional fluorescence microscope equipped with a cooled analog monochrome CCD camera (Model C 5985, Hamamatsu Photonics, Billerica, MA) and recorded using off the shelf personal computer hardware and software. Contrary to general belief, paraffin-embedded tissue sections do not show excessive nonspecific fluorescence. 

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