Immunofluorescence sectioning microscop

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. 

Direct fluorescence observations of sperm chromatin decondensation, MV binding to chromatin, and fusion processes in the sea urchin system have been performed with an upright Zeiss Standard microscope equipped with epifluores-cence. Chromatin decondensation is routinely monitored under a 40x, 0.75-mm NA, Zeiss objective, and nuclear envelope assembly under a Zeiss Neofluar lOOx, 1.3-mm NA, oil-immersion objective. Indirect immunofluorescence observations are performed using the same equipment. The microscopie should be fitted with appropriate fluorescence filter sets for the dyes used as described in Section V,A,1. 

Fig. 3. Confocal immunofluorescence showing RP2 localization in photoreceptors of the human retina. An 80-pm Vibratome section of human retina stained with rabbit hRP2-337-350 antiserum was detected with Cy3 conjugated donkey anti-rabbit and visualized by optical sectioning using a Zeiss LSM510 confocal microscope. OPL outer plexiform layer ONL outer nuclear layer OLM outer limiting membrane IS inner segment OS outer segment. Scale bar is 10 pm.

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