Histological studies of tissues and organs perfused with fluorescent microspheres may require modification of tissue processing methods in order to preserve the fluorescent microspheres. For example, formalin fixation followed by routine embedding in paraffin is not feasible as organic solvents (e.g., toluene) used during tissue processing dissolve polystyrene microspheres. The results of several different kinds of histological processing will be presented and the advantages and disadvantages of each discussed. These include the following: 1) Rapid freezing followed by cryo-microtome sectioning; 2) air-dried lung tissue followed by vibratome sectioning; 3) formalin fixation followed by vibratome sectioning; 4) formalin fixation followed by embedding in methyl methacrylate; 5) formalin fixation followed by embedding in paraffin-7-10 µm sections provide histological detail and serial sections are easily obtained but routine procedures were modified to preserve the microspheres. We evaluated several antimedia that would allow wax infiltration of tissue along with preservation of microsphere fluorescence and found that Histoclear II was the best substitute for toluene. Two additional issues are staining of sections and mountants to apply cover glasses to slides.
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