Abstract
Purpose :
Serial block-face scanning electron microscopy (SBF-SEM) has made it possible to collect large 3D ultrastructural data sets. However, precise identification of cells and structures would benefit from the use of specific antibody labeling as used in immunofluorescence microscopy. Antibodies can now be tagged with nanogold particles which do not interfere with antibody penetration into tissues. The nanoparticles are made visible (larger) through post-labeling gold toning. While this technique has been applied to transmission electron microscopy studies, to our knowledge it has yet to be applied to whole-tissue SBF-SEM imaging. Here we describe a microwave-assisted fixation and labeling strategy that improves detection and identification of gold-labeled mouse corneal nerves using SBF-SEM.
Methods :
Rabbit anti-mouse GAP43 (nerve proliferation factor) 1° antibody was used, with goat anti-rabbit nanogold (1.5 nm) 2° antibody. C57BL/6J mouse eyes were fixed in 1% paraformaldehyde and prepared in one of two ways: 1) 45 minute fixation and overnight antibody application, 2) microwave-assisted fixation and microwave-assisted antibody labeling. Microwaving occurred in an inverter microwave, augmented with a water-cooled plate, at 150 watts. Total microwave-assisted fixation time was 6 minutes, while total antibody labeling time was 32 minutes per antibody. Tissue was then post-fixed in 2.5% glutaraldehyde, gold-toned for 15 minutes and processed for SBF-SEM. Counting squares were used to ascertain the extent of labeling across the cornea. A Student’s t-test was used to compare signal-to-noise ratios.
Results :
The level of corneal nerve labeling was not significantly different between methods. Non-specific labeling of keratocytes, epithelial surface, and basal epithelium was significantly reduced using microwave-assisted labeling (P<0.001). The average signal-to-noise ratio for corneal nerve detection using microwave assisted labeling was 3-fold greater compared to standard non-microwave conditions.
Conclusions :
Fixation and immunogold-labeling using the microwave technique is an appropriate strategy for SBF-SEM imaging and cell identification. With respect to the latter, the gold particles facilitate accurate segmentation and 3D reconstruction from serial images. We anticipate this technique will prove useful in SBF-SEM studies of other tissues beyond the mouse cornea.
This is a 2020 Imaging in the Eye Conference abstract.