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 sm all intestine of mice fixed with 4% paraformaldehyde (4 P) or 0.5% par aformaldehyde and 15% of saturated picric acid (PPa). Before immunosta ining for laminin and tenascin, each specimen was pretreated with deox ycholate, while some 4 P-fixed specimens received further pepsin pretr eatment. Regardless of the fixatives employed, laminin and tenascin sh owed adequate immunofluorescence, Without pepsin pretreatment, the 4P- fixed specimens emitted conspicuous background fluorescence, and immun ofluorescence was weak in the lamina propria. Pepsin pretreatment redu ced the background fluorescence, but also diminished the immunofluores cence, especially that of tenascin. The PPa-fixed specimens displayed intense immunofluorescence of laminin and tenascin with very little ba ckground, even deeply within the lamina propria. When the PPa-fixed sp ecimens were immunostained for vasoactive intestinal peptide, immunopo sitive nerve fibres were observed within the lamina propria. Ultrastru ctural investigation of the PPa-fixed specimens revealed that membrano us structures in all cells were almost lost while tissue architecture was well preserved. These results indicate that PPa fixation and pretr eatment with deoxycholate are suitable for preparing whole mount speci mens for CLSM studies.