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A note on the preparation of whole mount samples suitable for observation with the confocal laser scanning microscope
L M Lee1, H Hashimoto, M Kusakabe
1Department of Anatomy, Jikei University School of Medicine, Tokyo, Japan.
Abstract:
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.
Insights
Picric acid and paraformaldehyde (PPa) fixation enhances immunofluorescence in mouse intestinal villi whole mounts for confocal laser scanning microscopy (CLSM). This method provides intense signals with minimal background, preserving tissue architecture for detailed analysis.
Area of Science:
- Microscopy and Imaging
- Cell Biology
- Histology
Background:
- Confocal laser scanning microscopy (CLSM) requires optimal specimen preparation for high-resolution imaging.
- Immunofluorescence techniques are crucial for visualizing specific proteins and structures in biological tissues.
- Whole mount preparations offer advantages for studying tissue architecture but present challenges in achieving uniform signal penetration and minimizing background fluorescence.
Purpose of the Study:
- To evaluate the impact of different fixatives and pretreatment methods on immunofluorescence quality in whole mount mouse intestinal villi for CLSM.
- To determine the optimal fixation and pretreatment protocol for enhancing signal-to-noise ratio and preserving tissue integrity.
Main Methods:
- Mouse proximal small intestine villi were fixed using 4% paraformaldehyde (4P) or 0.5% paraformaldehyde with 15% picric acid (PPa).
- Specimens underwent pretreatment with deoxycholate, with some 4P-fixed samples also receiving pepsin pretreatment.
- Immunostaining was performed for laminin, tenascin, and vasoactive intestinal peptide, followed by CLSM analysis.
- Ultrastructural examination was conducted to assess tissue preservation.
Main Results:
- PPa fixation yielded intense immunofluorescence for laminin and tenascin with minimal background, even in the lamina propria.
- 4P fixation without pepsin resulted in high background fluorescence and weak signals in the lamina propria.
- Pepsin pretreatment on 4P-fixed samples reduced background but also diminished immunofluorescence, particularly for tenascin.
- PPa-fixed specimens showed well-preserved tissue architecture, although some membranous structures were lost.
- Immunopositive nerve fibers were detected in the lamina propria of PPa-fixed specimens.
Conclusions:
- Picric acid and paraformaldehyde (PPa) fixation combined with deoxycholate pretreatment is superior for whole mount intestinal villi CLSM studies.
- This method significantly enhances immunofluorescence intensity and reduces background, facilitating detailed imaging of structures within the lamina propria.
- The PPa fixation protocol preserves overall tissue architecture, making it suitable for ultrastructural and immunofluorescence analyses.