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

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.