A note on the preparation of whole mount samples suitable for observation with the confocal laser scanning microscope.

The effects of fixatives and pretreatment on the immunofluorescence of whole mount specimens prepared for confocal laser scanning microscopy (CLSM) were examined. Intact villi were obtained from the proximal small intestine of mice fixed with 4% paraformaldehyde (4P) or 0.5% paraformaldehyde and 15% of saturated picric acid (PPa). Before immunostaining for laminin and tenascin, each specimen was pretreated with deoxycholate, while some 4P-fixed specimens received further pepsin pretreatment. Regardless of the fixatives employed, laminin and tenascin showed adequate immunofluorescence. Without pepsin pretreatment, the 4P-fixed specimens emitted conspicuous background fluorescence, and immunofluorescence was weak in the lamina propria. Pepsin pretreatment reduced the background fluorescence, but also diminished the immunofluorescence, especially that of tenascin. The PPa-fixed specimens displayed intense immunofluorescence of laminin and tenascin with very little background, even deeply within the lamina propria. When the PPa-fixed specimens were immunostained for vasoactive intestinal peptide, immunopositive nerve fibres were observed within the lamina propria. Ultrastructural investigation of the PPa-fixed specimens revealed that membranous structures in all cells were almost lost while tissue architecture was well preserved. These results indicate that PPa fixation and pretreatment with deoxycholate are suitable for preparing whole mount specimens for CLSM studies.